DNA typing by mass spectrometry with polymorphic DNA repeat markers

ABSTRACT

The present invention is related to the fields of genetic mapping and genetic identity detection, including forensic identification and paternity testing. This invention is more specifically directed to the use of mass spectrometry to detect length variation in DNA nucleotide sequence repeats (including variants of common alleles), such as microsatellites and short tandem repeats, and to DNA sequences provided as primers for the analysis of DNA tandem nucleotide repeat polymorphisms at specific loci on specific chromosomes.

The U.S. government may own rights in the present invention pursuant toGrant No. # 97-LB-VX-0003 from the U.S. National Institute of Justiceand cooperative agreement # 70NANB5H1029 from the U.S. Department ofCommerce.

This application claims benefit of Provisional Application No.60/059,415 filed Sep. 19, 1997.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The present invention is generally directed to the field of geneticidentity detection including forensic identification and paternitytesting as well as genetic mapping. The present invention is morespecifically directed to the use of mass spectrometry to detect lengthvariations in DNA nucleotide sequence repeats, often referred to asshort tandem repeats ("STR"), microsatellite repeats or simple sequencerepeats ("SSR"). The invention is also directed to DNA sequencesprovided for the analysis of STR polymorphisms at specific loci onspecific chromosomes.

B. Description of Related Art

Polymorphic DNA tandem repeat loci are useful DNA markers for paternitytesting, human identification, and genetic mapping. Higher organisms,including plants, animals and humans, contain segments of DNA sequencewith variable sequence repeats. Commonly sized repeats includedinucleotides, trinucleotides, tetranucleotides and larger. The numberof repeats occurring at a particular genetic locus vary depending on thelocus and the individual from a few to hundreds. The sequence and basecomposition of repeats can vary significantly, not even remainingconstant within a particular nucleotide repeat locus. DNA nucleotiderepeats are known by several different names including microsatelliterepeats, simple sequence repeats, short tandem repeats and variablenucleotide tandem repeats. As used herein, the term "DNA tandemnucleotide repeat" ("DTNR") refers to all types of tandem repeatsequences.

Thousands of DTNR loci have been identified in the human genome and havebeen predicted to occur as frequently as once every 15 kb. Populationstudies have been undertaken on dozens of these STR markers as well asextensive validation studies in forensic laboratories. Specific primersequences located in the regions flanking the DNA tandem repeat regionhave been used to amplify alleles from DTNR loci via the polymerasechain reaction ("PCR™"). Thus, the PCR™ products include the polymorphicrepeat regions, which vary in length depending on the number of repeatsor partial repeats, and the flanking regions, which are typically ofconstant length and sequence between samples.

The number of repeats present for a particular individual at aparticular locus is described as the allele value for the locus. Becausemost chromosomes are present in pairs, PCR™ amplifications of a singlelocus commonly yields two different sized PCR™ products representing twodifferent repeat numbers or allele values. The range of possible repeatnumbers for a given locus, determined through experimental sampling ofthe population, is defined as the allele range, and may vary for eachlocus, e.g., 7 to 15 alleles. The allele PCR™ product size range (allelesize range) for a given locus is defined by the placement of the twoPCR™ primers relative to the repeat region and the allele range. Thesequences in regions flanking each locus must be fairly conserved inorder for the primers to anneal effectively and initiate PCR™amplificattion. For purposes of genetic analysis di-, tri-, andtetranucleotide repeats in the range of 5 to 50 are typically utilizedin screens.

Many different primers have been designed for various DTNR loci andreported in the literature. These primers anneal to DNA sequencesoutside the DNA tandem repeat region to produce PCR™ products usually inthe size range of 100-800 bp. These primers were designed withpolyacrylamide gel electrophoretic separation in mind, because DNAseparations have traditionally been performed by slab gel or capillaryelectrophoresis. However, with a mass spectrometry approach to DTNRtyping and analysis, examining smaller DNA oligomers is advantageousbecause the sensitivity of detection and mass resolution are superiorwith smaller DNA oligomers.

The advantages of using mass spectrometry for characterizing DTNRsinclude a dramatic increase in both the speed of analysis (a few secondsper sample) and the accuracy of direct mass measurements. In contrast,electrophoretic methods require significantly longer lengths of time(minutes to hours) and can only measure the size of DTNRs as a functionof relative mobility to comigrating standards. Gel-based separationsystems also suffer from a number of artifacts that reduce the accuracyof size measurements. These mobility artifacts are related to thespecific sequences of DNA fragments and the persistence of secondary andtertiary structural elements even under highly denaturing conditions.

The inventors have performed significant work in developingtime-of-flight mass spectrometry ("TOF-MS") as a means for separatingand sizing DNA molecules, although other forms of mass spectrometry canbe used and are within the scope of this invention. Balancing thethroughput and high mass accuracy advantages of TOF-MS is the limitedsize range for which the accuracy and resolution necessary forcharacterizing DTNRs by mass spectrometry is available. Current state ofthe art for TOF-MS offers single nucleotide resolution up to ˜100nucleotides in size and four nucleotide resolution up to ˜160nucleotides in size. These numbers are expected to grow as newimprovements are developed in the mass spectrometric field.

Existing gel-based protocols for the analysis of DTNRs do not work withTOF-MS because the allele PCR™ product size range, typically between 100and 800 nucleotides, is outside the current resolution capabilities ofTOF-MS. Application of DTNR analysis to TOF-MS requires the developmentof new primer sets that produce small PCR™ products 50 to 160nucleotides in length, preferably 50 to 100 nucleotides in length.Amplified DNA may also be used to generate single stranded DNA productsthat are in the preferred size range for TOF-MS analysis by extending aprimer in the presence of a chain termination reagent. A typical classof chain termination reagent commonly used by those of skill in the artis the dideoxynucleotide triphosphates. Again, application of DTNRanalysis to TOF-MS requires that the primer be extended to generateproducts of 50 to 160 nucleotides in size, and preferably 50 to 100nucleotides in length.

Gel-based systems are capable of multiplexing the analysis of 2 or moreDTNR loci using two approaches. The first approach is to size partitionthe different PCR™ product loci. Size partitioning involves designingthe PCR™ primers used to amplify different loci so that that the allelePCR™ product size range for each locus covers a different and separablepart of the gel size spectrum. As an example, the PCR™ primers for LocusA might be designed so that the allele size range is from 250 to 300nucleotides, while the primers for Locus B are designed to produce anallele size range from 340 to 410 nucleotides.

The second approach to multiplexing 2 or more DTNR loci on gel-basedsystems is the use of spectroscopic partitioning. Current state of theart for gel-based systems involves the use of fluorescent dyes asspecific spectroscopic markers for different PCR™ amplified loci.Different chromophores that emit light at different color wavelengthsprovide the means for differential detection of two different PCR™products even if they are exactly the same size, thus 2 or more loci canproduce PCR™ products with allele size ranges that overlap. For example,Locus A with a green fluorescent tag produces an allele size range from250 to 300 nucleotides, while Locus B with a red fluorescent tagproduces an allele size range of 270 to 330 nucleotides. A scanning,laser-excited fluorescence detection device monitors the wavelength ofemissions and assigns different PCR™ product sizes, and theircorresponding allele values, to their specific loci based on theirfluorescent color.

In contrast, mass spectrometry directly detects the molecule preventingthe use of optical spectroscopic partitioning as a means formultiplexing. While it is possible to have a limited use of sizepartitioning with TOF-MS, the limited size range of high-resolutiondetection by TOF-MS makes it likely that only 2 different loci can bemultiplexed and size partitioned. In many cases, it may not be possibleto even multiplex 2 loci and maintain a partitioning of the 2 differentallele size ranges. Therefore, new methods are needed in order to employmass spectrometry for the analysis of multiplexed DTNRs.

SUMMARY OF THE INVENTION

It is, therefore, a goal of the present invention to provide newlydesigned PCR™ primers which are closer to the repeat regions then havepreviously been employed providing for the efficient analysis by TOF-MS.Specifically, the invention provides oligonucleotide primers designed tocharacterize various DTNR markers useful for human identity testing. Theprimers are for use in PCR™ amplification schemes, however, one of skillin the art could, in light of the present disclosure, employ them togenerate appropriate size nucleic acid products for TOF-MS analysisusing other methods of extending one or more of the disclosed primers.Additionally, these primers and their extension products are suitablefor detection by mass spectrometry. Thus, applications of this inventioninclude forensic and paternity testing and genetic mapping studies.

An embodiment of the present invention encompasses an oligonucleotideprimer for use in analyzing alleles of a DNA tandem nucleotide repeat ata DNA tandem nucleotide repeat locus by mass spectrometry, whichincludes a nucleotide sequence that contains a flanking region of thelocus where the primer upon extension generates a product that iscapable of being analyzed by mass spectrometry. Preferably, theoligonucleotide primer's 3' end will be complementary to a regionflanking a DNA tandem repeat region immediately adjacent to the DNAtandem repeat region or may further extend up to one, two, three, fouror five tandem repeats into the DNA tandem repeat region. Used in thiscontext "immediately adjacent" or "immediately flanking" means one, two,three, or four nucleotides away from the DNA tandem repeat region of theDNA tandem repeat locus.

The oligonucleotide primers of this invention are designed to generateextension products amenable to mass spectral analysis and containing aDTNR sequence, or region of interest, for which one is interested indetermining the mass. The "flanking" regions of a DTNR locus are theportions of DNA sequence on either side of the DTNR region of interest.For embodiments employing PCR™ primers and polymerases to amplify a DTNRsequence, the primers are sufficiently complementary to a portion of oneor more flanking regions of the DTNR locus to allow the primer toeffectively anneal to the target nucleic acid and provide a site toextend a complement to the target nucleic acid via PCR™. For embodimentsemploying primer extension, a preferred method is to use a single primerthat is sufficiently complementary to allow effective anealling to aportion of a target DTNR locus flanking region in conjunction with achain termination reagent. The chain termination reagent allows theproduction of discreet limited size nucleic acid products for massspectral analysis. Preferred chain termination reagent for use in thepresent invention are dideoxynucleotide triphosphates. Therefore, forthe methods comprising any type of primer extension, it is preferredthat at least one of the primers is sufficiently complementary to aportion of a flanking region that is preferably adjacent to or close tothe DTNR region of interest, generally within about 40 nucleotides ofthe DNA tandem nucleotide repeat region. As used in this context,"about" means anywhere from ±1 to 40 nucleotides, and all the integersin between, for example, ±1, ±2, ±3, ±4, ±5, ±6, ±7, ±8, ±9, ±10, etc.nucleotides.

The primer extension products are preferably single-stranded and may beany size that can be adequately resolved by mass spectrometric analysis.Preferably, detected, the final product single-stranded target nucleicacids are less than about 160 or 150 bases in length. More preferably,the extended nucleic acid products are from about 10 to 100 or 120 basesin length. As used in this context, "about" means anywhere from ±1 to 20bases, and all the integers in between, for example, ±1, ±2, ±3, ±4, ±5,±6, ±7, ±8, ±9, ±10, etc. bases.

As used herein "a" will be understood to mean one or more. Thus, "a DNAtandem repeat marker" may refer, for example, to one, two, three, four,five or more DNA tandem repeat markers.

The present invention is also directed to new oligonucleotide primerswhich have been designed to match a portion of the flanking regions forvarious DTNR loci. Specific embodiments of this invention includeoligonucleotide primers designed to amplify the following DTNR loci:CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51,D21S11, DYS19, F13A1, FES/FPS, FGA, HPRTB, TH01, TPOX, DYS388, DYS391,DYS392, DYS393, D2S1391, D18S535, D2S1338, D19S433, D6S477, D1S518,D14S306, D22S684, F13B, CD4, D12S391, D10S220 and D7S523. With theexception of D3 S1358, sequences for the STR loci of this invention areaccessible to the general public through GenBank using the accessionnumbers listed in Table 1. These oligonucleotide primers may preferablycontain a cleavable site, such as a recognition site for Type II and IISrestriction endonucleases, an exonuclease blocking site, or a chemicallycleavable site, for reducing the length of the amplified product andincreasing the mass spectral resolution.

Examples of some oligonucleotide primers that may be employed foramplifying these loci are listed in SEQ ID NO:1 through SEQ ID NO:103.Preferred oligonucleotide primers that also contain a cleavablephosphorothioate linkage and biotin moiety for immobilization on anavidin, streptavidin solid support are sequences according to SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87,SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92,SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97,SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:103. These newlydesigned primers generate nucleic acid extension products which aresmaller than those used previously with electrophoresis separationmethods. Additionally, these primers may be used in other methods ofprimer extension known to those of skill in the art.

It will be apparent to one skilled in the art that some variations ofthese primers will also serve effectively, for example, adding ordeleting one or a few bases from the primer and/or shifting the positionof the primer relative to the DTNR sequence by one or a few bases. Thus,primers encompassed by the present invention include the primersspecifically listed as well as modifications of these primers. Althoughthese sequences are all biotinylated at the 5' end and contain aphosphorothioate linkage at a particular location, one of skill in theart would recognize that similar primers having biotin moieties and thecleavable groups at other sites would also be encompassed by the presentinvention. Primers containing types of immobilization attachments sitesother than biotin, for example, would also be encompassed. Typically,the placement of the cleavable group is not critical as long as it isclose enough to the 3' end to cleave the cleave the nucleic acidextension product to a reduced-length amplified product that is amenableto mass spectral analysis. These primers in pairs may also be combinedto generate overlapping PCR™ product sizes which are all distinguishableby mass. However, for embodiments multiplexing multiple DTNR loci withoverlapping allelic mass ranges, strategic placement of the cleavablegroup may effect a separation or an interleaving of mass spectral peaks.

Another embodiment of this invention encompasses a kit for analyzingalleles of a DTNR locus in a target nucleic acid, having a first strandand a second complementary strand, by mass spectrometry which includes afirst primer complementary to the flanking region of a DNA tandemnucleotide repeat region and a second primer complementary to theopposite flanking region of a DNA tandem nucleotide repeat region.Preferred kits of this invention are kits for analyzing the followingDTNR loci: CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539,D18S51, D21S11, DYS19, F13A1, FES/FPS, FGA, HPRTB, TH01, TPOX, DYS388,DYS391, DYS392, DYS393, D2S1391, D18S535, D2S1338, D19S433, D6S477,D1S518, D14S306, D22S684, F13B, CD4, D12S391, D10S220 and D7S523.

Another embodiment of this invention encompasses a kit for analyzingalleles of a multiple DTNR loci in a target nucleic acid by massspectrometry, which includes a plurality of primers complementary to theflanking regions of DNA tandem nucleotide repeat regions. Preferred kitsof this invention are kits for analyzing the following DTNR loci:CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51,D21S11, DYS19, F13A1, FES/FPS, FGA, HPRTB, TH01, TPOX, DYS388, DYS391,DYS392, DYS393, D2S1391, D18S535, D2S1338, D19S433, D6S477, D1S518,D14S306, D22S684, F13B, CD4, D12S391, D10S220 and D7S523.

The primers employed with these kits may preferably have cleavablesites, such as a recognition site for a restriction endonuclease, anexonuclease blocking site, or a chemically cleavable site. Preferredchemically cleavable sites encompass modified bases, modified sugars(e.g., ribose), and chemically cleavable groups incorporated into thephosphate backbone, such as dialkoxysilane, 3'-(S)-phosphorothioate,5'-(S)-phosphorothioate, 3'-(N)-phosphoroamidate, or5'-(N)-phosphoroamidate linkages. Another preferred embodiment is a kitemploying a first primer that is capable of attaching to a solidsupport.

For primer extension by PCR amplification, it is preferable to employthese primers in pairs. Preferred pairs of primers include thefollowing: a sequence according to SEQ ID NO:1 and a sequence accordingto SEQ ID NO:2; a sequence according to SEQ ID NO:3 and a sequenceaccording to SEQ ID NO:4; a sequence according to SEQ ID NO:5 and asequence according to SEQ ID NO:6; a sequence according to SEQ ID NO:7and a sequence according to SEQ ID NO:8; a sequence according to SEQ IDNO:9 and a sequence according to SEQ ID NO:10; a sequence according toSEQ ID NO:11 and a sequence according to SEQ ID NO:12; a sequenceaccording to SEQ ID NO:13 and a sequence according to SEQ ID NO:14; asequence according to SEQ ID NO:15 and a sequence according to SEQ IDNO:16; a sequence according to SEQ ID NO:17 and a sequence according toSEQ ID NO:18; a sequence according to SEQ ID NO:19 and a sequenceaccording to SEQ ID NO:20; a sequence according to SEQ ID NO:21 and asequence according to SEQ ID NO:22; a sequence according to SEQ ID NO:23and a sequence according to SEQ ID NO:24; a sequence according to SEQ IDNO:25 and a sequence according to SEQ ID NO:26; a sequence according toSEQ ID NO:27 and a sequence according to SEQ ID NO:28; a sequenceaccording to SEQ ID NO:29 and a sequence according to SEQ ID NO:30; asequence according to SEQ ID NO:31 and a sequence according to SEQ IDNO:32; a sequence according to SEQ ID NO:49 and a sequence according toSEQ ID NO:83; a sequence according to SEQ ID NO:52 and a sequenceaccording to SEQ ID NO:84; a sequence according to SEQ ID NO:54 and asequence according to SEQ ID NO:85; a sequence according to SEQ ID NO:56and a sequence according to SEQ ID NO:86; a sequence according to SEQ IDNO:58 and a sequence according to SEQ ID NO:87; a sequence according toSEQ ID NO:59 and a sequence according to SEQ ID NO:88; a sequenceaccording to SEQ ID NO:62 and a sequence according to SEQ ID NO:89; asequence according to SEQ ID NO:63 and a sequence according to SEQ IDNO:90; a sequence according to SEQ ID NO:66 and a sequence according toSEQ ID NO:91; a sequence according to SEQ ID NO:67 and a sequenceaccording to SEQ ID NO:92; a sequence according to SEQ ID NO:70 and asequence according to SEQ ID NO:93; a sequence according to SEQ ID NO:72and a sequence according to SEQ ID NO:94; a sequence according to SEQ IDNO:74 and a sequence according to SEQ ID NO:95; a sequence according toSEQ ID NO:76 and a sequence according to SEQ ID NO:96; a sequenceaccording to SEQ ID NO:78 and a sequence according to SEQ ID NO:97; asequence according to SEQ ID NO:80 and a sequence according to SEQ IDNO:98; a sequence according to SEQ ID NO:66 and a sequence according toSEQ ID NO:99; a sequence according to SEQ ID NO:33 and a sequenceaccording to SEQ ID NO:100; and a sequence according to SEQ ID NO:101and a sequence according to SEQ ID NO:103.

In one embodiment, at least one of the primers used to prepare thenucleic acid extension product contains a surface binding moiety, suchas a biotin moiety, at the 5'-end and a cleavable moiety, such as aphosphorothioate linkage (see FIGS. 7A and 7B), near the 3'-end for acapture and release assay, such as one using streptavidin-coatedmagnetic beads for binding biotinylated primers, described in PCT PatentApplication No. WO 96/37630, and incorporated herein by reference. Theselinkages are often referred as thiophosphate linkages as well.Incorporation of a method for obtaining single-stranded PCR™ products,such as is possible with the primer modifications described above, ispreferred. Removal of one of the two strands halves the number of DNAoligomers that will be visualized by TOF-MS and improves the likelihoodof resolving all PCR™ product strands.

Another embodiment of this invention encompasses a method for analyzingDNA tandem nucleotide repeat alleles at a DNA tandem nucleotide repeatlocus in a target nucleic acid by mass spectrometry which includes thesteps of a) obtaining a target nucleic acid containing a DNA tandemnucleotide repeat region; b) extending the target nucleic acid using oneor more primers to obtain a limited size range of nucleic acid extensionproducts, wherein the primers are complementary to a sequence flankingthe DNA tandem nucleotide repeat of said locus; and c) determining themass of the nucleic acid extension products by mass spectrometry, wherethe target nucleic acid is normally double-stranded (i.e. it has a firststrand and a second complementary strand). Nucleic acid extensionproducts may be generated in this method by any means known to those ofskill in the art, and particularly either by amplification, such as PCRamplification, or by primer extension in conjunction with a chaintermination reagent. Preferred primers may immediately flank the DNAtandem repeat locus, or may further extend up to one, two, three, fouror five tandem repeats into the DNA tandem repeat region. Used in thiscontext "immediately adjacent" or "immediately flanking" means one, two,three, or four nucleotides away from the DNA tandem repeat region of theDNA tandem repeat locus. Preferred primers may contain a cleavable site,such as a recognition site for a restriction endonuclease, anexonuclease blocking site, or a chemically cleavable site, and becapable of attaching to a solid support.

These primers may be capable of directly or indirectly attaching to asolid support via covalent or noncovalent binding. The primers maycontain an immobilization attachment site (IAS) for attachment to asolid support. This site is usually upstream of the chemically cleavablesite. A suitable immobilization attachment site is any site capable ofbeing attached to a group on a solid support. These sites may be asubstituent on a base or sugar of the primer. An IAS may be, forexample, an antigen, biotin, or digoxigenin. This attachment allows forisolation of only one strand of an amplified product. Such isolation ofeither single-stranded or double-stranded amplified target nucleic acidsgenerally occurs prior to the application of the nucleic acids to thematrix solution, resulting in well-defined mass spectral peaks andenhanced mass accuracy. The matrix solution can be any of the knownmatrix solutions used for mass spectrometric analysis, including3-hydroxypicolinic acid ("3-HPA"), nicotinic acid, picolinic acid,2,5-dihydroxybenzoic acid, and nitrophenol.

For example, in one embodiment, a strand of a target nucleic acidextension product may be bound or attached to a solid support to permitrigorous washing and concomitant removal of salt adducts, unwantedoligonucleotides and enzymes. Either a double-stranded or asingle-stranded nucleic acid extension product may be isolated for massspectrometric analysis. The single-stranded target nucleic acidextension product analyzed by MS may be either the strand bound or notbound to the solid support.

When an unbound strand is used for MS analysis, it is typically purifiedby first washing the bound strand and its attached complement underconditions not sufficiently rigorous to disrupt the strand's attachmentto its bound complement. After unwanted biomolecules and salts areremoved, the complement may then be released under more rigorousconditions. In contrast, when the bound strand is to be analyzed, it istypically washed under more vigorous conditions such that theinteractions between the bound strand, if present, and its unboundcomplement is disrupted. This allows the unbound strand to be washedaway with the other salts and unwanted biomolecules. Cleavable linkersor cleavable primers may be used to release the bound strand from thesolid support prior to MS analysis.

Preferred primers for practicing this method include primers designed toamplify DTNR loci selected from the group consisting of CSF1PO, D3S1358,D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, DYS19, F13A1,FES/FPS, FGA, HPRTB, TH01, TPOX, DYS388, DYS391, DYS392, DYS393,D2S1391, D18S535, D2S1338, D19S433, D6S477, D1S518, D14S306, D22S684,F13B, CD4, D12S391, D10S220 and D7S523. Preferred pairs of primersdesigned to amplify these loci include: a sequence according to SEQ IDNO:1 and a sequence according to SEQ ID NO:2; a sequence according toSEQ ID NO:3 and a sequence according to SEQ ID NO:4; a sequenceaccording to SEQ ID NO:5 and a sequence according to SEQ ID NO:6; asequence according to SEQ ID NO:7 and a sequence according to SEQ IDNO:8; a sequence according to SEQ ID NO:9 and a sequence according toSEQ ID NO:10; a sequence according to SEQ ID NO:11 and a sequenceaccording to SEQ ID NO:12; a sequence according to SEQ ID NO:13 and asequence according to SEQ ID NO:14; a sequence according to SEQ ID NO:15and a sequence according to SEQ ID NO:16; a sequence according to SEQ IDNO:17 and a sequence according to SEQ ID NO:18; a sequence according toSEQ ID NO:19 and a sequence according to SEQ ID NO:20; a sequenceaccording to SEQ ID NO:21 and a sequence according to SEQ ID NO:22; asequence according to SEQ ID NO:23 and a sequence according to SEQ IDNO:24; a sequence according to SEQ ID NO:25 and a sequence according toSEQ ID NO:26; a sequence according to SEQ ID NO:27 and a sequenceaccording to SEQ ID NO:28; a sequence according to SEQ ID NO:29 and asequence according to SEQ ID NO:30; a sequence according to SEQ ID NO:31and a sequence according to SEQ ID NO:32; a sequence according to SEQ IDNO:49 and a sequence according to SEQ ID NO:83; a sequence according toSEQ ID NO:52 and a sequence according to SEQ ID NO:84; a sequenceaccording to SEQ ID NO:54 and a sequence according to SEQ ID NO:85; asequence according to SEQ ID NO:56 and a sequence according to SEQ IDNO:86; a sequence according to SEQ ID NO:58 and a sequence according toSEQ ID NO:87; a sequence according to SEQ ID NO:59 and a sequenceaccording to SEQ ID NO:88; a sequence according to SEQ ID NO:62 and asequence according to SEQ ID NO:89; a sequence according to SEQ ID NO:63and a sequence according to SEQ ID NO:90; a sequence according to SEQ IDNO:66 and a sequence according to SEQ ID NO:91; a sequence according toSEQ ID NO:67 and a sequence according to SEQ ID NO:92; a sequenceaccording to SEQ ID NO:70 and a sequence according to SEQ ID NO:93; asequence according to SEQ ID NO:72 and a sequence according to SEQ IDNO:94; a sequence according to SEQ ID NO:74 and a sequence according toSEQ ID NO:95; a sequence according to SEQ ID NO:76 and a sequenceaccording to SEQ ID NO:96; a sequence according to SEQ ID NO:78 and asequence according to SEQ ID NO:97; a sequence according to SEQ ID NO:80and a sequence according to SEQ ID NO:98; a sequence according to SEQ IDNO:66 and a sequence according to SEQ ID NO:99; a sequence according toSEQ ID NO:33 and a sequence according to SEQ ID NO:100; and a sequenceaccording to SEQ ID NO:101 and a sequence according to SEQ ID NO:103.

The present invention also focuses on an improved method of multiplexingthe analysis of nucleic acid extension products derived from DNAnucleotide repeat loci. This method differs from known methods ofmultiplexing DTNR analysis in that mass spectrometry is employed and therange of possible nucleic acid extension products for the multiplexedloci, the allele nucleic acid extension product size ranges, may bespecifically chosen to overlap in the mass scale yet be uniquelyresolved and detected.

Thus, this invention encompasses methods for analyzing more than onetarget nucleic acid in which the target nucleic acids are used toproduce more than one nucleic acid product extension product and whereeach nucleic acid extension product may comprise a different DTNRsequence. A preferred embodiment encompasses simultaneously determiningthe mass of more than one DNA tandem nucleotide repeat allele at morethan one DNA tandem nucleotide repeat loci. According to this embodimentseveral amplification products containing various DTNR sequences fromdifferent DTNR loci may be analyzed in the same solution and spectrum.

Additionally, the DNA tandem nucleotide repeat loci may have overlappingallelic mass ranges (see FIGS. 4 and 5). The term "overlapping allelicmass ranges" is defined to mean that the alleles that may be present fora particular DTNR locus have masses that overlap, or coincide, asobserved by mass spectrometry with the masses for alleles from anotherDTNR locus. The methods of the present invention allow one to resolvethese alleles by mass spectrometry either by increasing the massseparation of these peaks or by modifying the mass of the amplifiedproducts containing the various DTNR sequences such that theamplification products have interleaving mass spectral peaks (see FIG.6).

This novel interleaved multiplexing approach overcomes the TOF-MSlimitations for size partitioning and takes advantage of the high massaccuracy of the method within the high resolution mass range below about160 nucleotides in size. One specific embodiment encompasses a methodthat involves the design of specific primer or primers that producenucleic acid extension products for a first locus with defined allelemass values. The primer or primers for second locus are then selected sothat while the mass range for the predicted nucleic acid extensionproducts of the primers overlap with the mass range for the products ofthe first locus, the specific predicted nucleic acid extension productmass values differ from those of the first locus and therefore can beuniquely resolved by TOF-MS. Further loci may be added to the multiplexusing the same method such that three, four, five, six, seven, eight,nine, ten or more loci may be analyzed simultaneously.

The basic limits for this multiplexing are defined by the ability toresolve all possible nucleic acid extension products within a mixture.It is not inconceivable that as many as 10 different loci might beinterleaved and uniquely resolved. In addition to multiplexing two ormore DTNRs it is also possible to use this invention to interleavemixtures of DTNRs with specific nucleic acid extension products arisingfrom nonrepeat loci, e.g., a DTNR locus with allelic nucleic acidextension products 72, 76, 80, 84 and 88 nucleotides in size could besimultaneously analyzed with a nucleic acid extension product 82nucleotides in size.

The ability to interleave loci requires that thenucleic acid extensionproduct mass values for all possible allele values should preferably beknown. These allele mass values may be determined empirically or morelikely by calculation using the known loci sequences. In many cases itmay be necessary to "fine tune" the allele mass values for one or moreloci in a multiplexed mixture in order to prevent unresolvable overlapbetween two Nucleic acid extension products. For example, allele 5 forLocus A may be only 5 Da different in mass than allele 9 for Locus Bpreventing resolution of those two Nucleic acid extension products bymass spectrometry. Mass modifications to one or both loci may be used toincrease this mass difference to 100 Da.

Adjusting the allele mass values for any given locus may be done by anynumber of methods including: increasing or decreasing the size the ofthe nucleic acid extension products via altered sequences and placementof the primers; addition of nonhybridizing nucleotides to the 5' ends ofone or more primers, addition of nonnucleotide chemical modificationsinternally or to the ends of one or both primers; alterations in basecomposition within one or both primers, including the use of nonstandardnucleotides, that may or may not result in mismatches within theprimers; incorporation of and specific placement of a chemicallycleavable moiety within the primer backbone to reduce the length of thenucleic acid extension product by a selected amount; enzymatic cleavageof the nucleic acid extension products using a restriction endonucleasethat recognizes a restriction site within one or both primers or withinthe nucleic acid extension product itself; use of a 5' to 3' exonucleasein concert with exonuclease blocking modified nucleotides containedwithin one or more primers; incorporation of nonstandarddeoxyribonucleotides or chemically or isotopically modified nucleotidesduring polymerization; any number of methods of mass modifying byaddition of chemical moieties post amplification; by using differentchain termination reagents in conjunction with primer extension; or anynumber of other means that anyone skilled in the art would be able toidentify.

Another embodiment encompasses a method of multiplexing amplificationproducts containing DTNRs having overlapping allelic ranges where atleast one amplification product contains a mass modified nucleotide.Mass modified nucleotides include nucleotides to which nonnucleotidemoieties have been chemically attached; bases having alteredcompositions; nonstandard nucleotides, that may or may not result inmismatches within the primers; and any bases whose masses have beenmodified through the addition of chemical moieties after theamplification step.

Alternatively, the length of at least one extension product may bereduced by cleaving the extension product at a cleavable site such as arestriction endonuclease cleavage site, an exonuclease blocking site, ora chemically cleavable site. Preferred chemically cleavable sites formultiplexing include modified bases, modified sugars (e.g., ribose), ora chemically cleavable group incorporated into the phosphate backbone,such as a dialkoxysilane, 3'-(S)-phosphorothioate,5'-(S)-phosphorothioate, 3'-(N)-phosphoroamidate, or5'-(N)-phosphoroamidate. Preferred primers may also be capable ofattaching to a solid support.

Another embodiment of this invention encompasses a method formultiplexing the detection of more than one amplified DNA tandemnucleotide repeat marker from more than one DNA tandem nucleotide repeatloci including: determining the mass of more than one nucleic acidextension product by mass spectrometry, where the DNA tandem nucleotiderepeat loci each comprise a DNA tandem repeat sequence and a flankingsequence and have overlapping allelic mass ranges. Typically, at leastone of the target nucleic acid extension products may contain a massmodifying group.

"Mass modifying groups" may comprise any group that alters the mass ofthe amplified products to produce interleaving or otherwise resolvablemass spectral peaks. These groups, which may be incorporated during orafter primer extension, may be mass modified nucleotides, nonstandarddeoxyribonucleotides, or even cleavable sites as cleaving such a sitemodifies the mass by reducing the length of the extension product. Asused in this context, modified or nonstandard bases are generallyunderstood to include bases not found in DTNR locus flanking the DTNRsequence of the sample or target nucleic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a mass spectrum of an allelic ladder from the tyrosinehydroxylase gene ("TH01"). Most of the common alleles for this STRmarker (alleles 5, 6, 7, 8, 9, 9.3, and 10) can be seen. Alleles 9.3 and10 differ by a single nucleotide while the other alleles are separatedby four bases.

FIG. 2 displays mass spectra for several samples from the TPOX locus.The top spectrum is an allelic ladder containing alleles ranging from 6to 13 repeats while the other spectra show the isolation of variousalleles for this locus.

FIG. 3A displays the mass spectrum for the CSF1PO locus.

FIG. 3B displays the mass spectrum for the D3S1358 locus.

FIG. 3C displays the mass spectrum for the D5S818 locus.

FIG. 3D displays the mass spectrum for the D7S820 locus.

FIG. 3E displays the mass spectrum for the D8S1179 locus.

FIG. 3F displays the mass spectrum for the D13S317 locus.

FIG. 3G displays the mass spectrum for the D16S539 locus.

FIG. 3H displays the mass spectrum for the D18S51 locus.

FIG. 31 displays the mass spectrum for the D21S11 locus.

FIG. 3J displays the mass spectrum for the DYS19 locus.

FIG. 3K displays the mass spectrum for the F13A1 locus.

FIG. 3L displays the mass spectrum for the FES/FPS locus.

FIG. 3M displays the mass spectrum for the FGA locus.

FIG. 3N displays the mass spectrum for the HPRTB locus.

FIG. 3O displays the mass spectrum for the TH01 locus.

FIG. 3P displays the mass spectrum for the TPOX locus.

FIG. 4 is a simulated multiplex STR analysis of alleles with overlappingsize ranges. This diagram depicts the expected masses for known allelesof TPOX and TH01.

FIG. 5 are mass spectra of mixtures of TH01 and TPOX allelic ladders.Using the primer sequences for TH01 (SEQ ID NO.:29 and SEQ ID NO.:30)and TPOX (SEQ ID NO.:31 and SEQ ID NO.:32), alleles between thedifferent STR systems differ by only 120 Daltons (top spectrum). Byadding two nucleotides to the 5'-end of the reverse primer for TPOX (SEQID NO.:32), the TPOX allele masses are increased by ˜600 Daltons, makingthem easier to resolve.

FIG. 6 is a simulated multiplex STR analysis depicting the expectedmasses for D16S539 and D7S820 known alleles. Even with different repeatsequences, all known alleles may be resolved by mass spectroscopy.

FIG. 7A shows the chemical formula for2'-deoxythymidine-3'-(S)-phosphorothioate.

FIG. 7B shows the chemical formula for2'-deoxythymidine-5'-(S)-phosphorothioate.

FIG. 8A shows the expected allele sizes for CTT multiplex analyses. TheCTT multiplex is directed to the three STR loci CSF1PO, TPOX, and TH01.

FIG. 8B illustrates the results of the analysis of a sample using theCTT multiplex. The sample is shown to contain a homozygous TPOX allele8, heterozygous TH01 alleles 6 and 9.3, and a homozygous CSF1PO allele12.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention focuses on a mass spectrometric method ofmultiplexing the analysis of Nucleic acid extension products whichoverlap in mass derived from DNA nucleotide repeat loci. For example, toresolve all possible alleles of the DTNRs being analyzed the masses ofthe Nucleic acid extension products from two or more DTNR markers may beoffset from one another so that any two possible alleles (or any twopossible common alleles) do not overlap in mass within the massresolution of the mass spectrometer, yet the ranges of the possiblealleles do overlap. Within the overlapping mass range, defined as themass range held is common by two loci with defined allele size ranges,the DTNR marker may be offset from one another by some fraction of themass of the sequence repeat unit, e.g. for tetranucleotide DTNR markersmass offsets less than four nucleotide, for dinucleotide DTNRs massoffsets less than 2 nucleotides. Other types of offset, such as may befound when multiplexing dinucleotide repeat loci with tetranucleotide orcomplex nucleotide repeat loci, will be apparent to one skilled in theart.

This approach overcomes the TOF-MS limitations for size partitioning,where the PCR™ product for the allele range of two or more sets ofpossible loci do not overlap, by taking advantage of the high massaccuracy associated with mass spectroscopy within the high resolutionmass range (below ˜160 nucleotides in size). Although this method iscurrently most useful for oligonucleotides below ˜160 nucleotides, thissize is a function of the number of nucleotides in the repeat as well asthe resolution of the mass spectroscopic method. Therefore, largeroligonucleotides are also useful with the present invention,particularly where larger repeat sequences (tetra- vs. dinucleotides) oras advances in mass spectroscopy allow for greater mass resolution inhigher mass ranges.

This multiplexing method involves the design of specific primers thatproduce Nucleic acid extension products for a first locus with definedallele mass values. The primers for the second locus are then chosen sothat while the mass range for the different alleles overlaps with themass range for the first locus, the specific allele mass values differfrom those of the first locus and therefore can be uniquely resolved byTOF-MS. The identity of each allele, defined by the specific Nucleicacid extension products being characterized, is uniquely determinedusing the high accuracy molecular mass values provided by TOF-MS. Incontrast, gel-based methods are not capable of providing accurate massvalues for uniquely identifying each product within a multiplexed,allelically interleaved mixture of Nucleic acid extension products. Thebasic limits for this multiplexing method are defined by the ability toresolve all possible, or all common, Nucleic acid extension productswithin a mixture. Potentially as many as 10 different loci might beinterleaved and fully resolved.

The invention further relates to primers designed to characterize 33 DNArepeat markers useful for human identity testing. Applications includeforensic and paternity testing as well as genetic mapping studies. TheseDTNR markers are useful in PCR™ amplification, preferably as pairs ofoligonucleotide primers, and in other methods of primer extension may beused as single primers, the extension products of which may beaccurately detected by mass spectrometry as they are smaller than thoseused previously with electrophoresis separation methods.

These new oligonucleotide primers are designed to match a portion of theflanking regions for DTNR loci consisting of: CSF1PO, D3S1358, D5S818,D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, DYS19, F13A1,FES/FPS, FGA, HPRTB, TH01, TPOX, DYS388, DYS391, DYS392, DYS393,D2S1391, D18S535, D2S1338, D19S433, D6S477, D1S518, D14S306, D22S684,F13B, CD4, D12S391, D10S220 a With the exception of D3S1358, sequencesfor the STR loci of this invention are accessible to the general publicthrough GenBank using the accession numbers listed in Table 1. Thesequence ID Numbers given in Table 1 correspond to the DNA sequence ofthe DNA tandem repeat regions of each locus and its flanking regions.Flanking sequences further from the DTNR region could easily be obtainedby one of skill in the art by accessing the GenBank listings. FIGS.3A-3P display mass spectra for each of the STR loci listed in TABLE 1.It will be apparent to one skilled in the art that small variation ofthese primers will also serve effectively, for example, adding ordeleting one or a few bases from the primer and/or shifting the positionrelative to the template sequence by one or a few bases.

The use of a hybridization probe of about 14-25 nucleotides in lengthallows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 14 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of 15 to 25 contiguous nucleotides,or even longer where desired.

Hybridization probes may be selected from any portion of any of thesequences disclosed herein. All that is required is to review the primersequences set forth in Table1 or to any continuous portion of thesequence as in the DTNR loci, whose locus sequence ID numbers are listedin Table 1 or any other DTNR locus, from about 14-25 nucleotides inlength up to and including the full length sequence, that one wishes toutilize as a probe or primer. The choice of probe and primer sequencesmay be governed by various factors known to those of skill in the art.

The process of selecting and preparing a nucleic acid segment thatincludes a contiguous sequence from within the DTNR loci, whose locussequence ID numbers are listed in Table 1 or any other DTNR locus, mayalternatively be described as preparing a nucleic acid fragment. Ofcourse, fragments may also be obtained by other techniques such as,e.g., by mechanical shearing or by restriction enzyme digestion. Smallnucleic acid segments or fragments may be readily prepared by, forexample, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCR™ technology of U.S. Pat. No. 4,683,202(incorporated herein by reference), by introducing selected sequencesinto recombinant vectors for recombinant production, and by otherrecombinant DNA techniques generally known to those of skill in the artof molecular biology.

Accordingly, the nucleotide sequences of the invention may be chosen fortheir ability to selectively form duplex molecules with complementarystretches of the flanking regions of DNA nucleotide repeat regions.Depending on the application envisioned, one will desire to employvarying conditions of hybridization to achieve varying degrees ofselectivity of probe towards target sequence. For applications requiringhigh selectivity, one will typically desire to employ relativelystringent conditions to form the hybrids, e.g., one will selectrelatively low salt and/or high temperature conditions, such as providedby a salt concentration of from about 0.02 M to about 0.15 M salt attemperatures of from about 50° C. to about 70° C. Such selectiveconditions tolerate little, if any, mismatch between the probe and thetemplate or target strand.

Of course, for some applications, less stringent (reduced stringency)hybridization conditions will be tolerated by the primer extensionsystem in order to allow sufficiently specific formation of theheteroduplex of primer and target DNA. In these circumstances, one maydesire to employ salt conditions such as those of from about 0.15 M toabout 0.9 M salt, at temperatures ranging from about 20° C. to about 55°C. Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulatedto ensure that a primer sequence will yield extension product mainlyfrom the desired target DTNR locus.

                                      TABLE 1                                     __________________________________________________________________________                                   Locus                                                                         SEQ ID                                         Primer                         No..sup.3                                      SEQ ID                   STR   (GenBank                                                                            PCR ™                                 No..sup.1                                                                         Primer Sequence (5'-3')                                                                            Locus.sup.2                                                                         Accession)                                                                          Size.sup.4                                                                          Repeat.sup.5                       __________________________________________________________________________    1, 100                                                                            ACAGTAACTGCCTTCATAGATAG                                                                            CSF1PO-F                                                                            104   12 = 113 bp                                                                         AGAT                               2, 33                                                                             GTGTCAGACCCTGTTCTAAGTA                                                                             CSF1PO-R                                                                            (X14720)                                       3   ACTGCAGTCCAATCTGGGT  D3S1358-F                                                                           --    16 = 109 bp                                                                         GAYA                               4, 34                                                                             ATGAAATCAACAGAGGCTTG D3S1358-R                                                                           --                                             5, 35                                                                             CTCTTGGTATCCTTATGTAATATT                                                                           D5S818-F                                                                            105   11 = 105 bp                                                                         AGAT                               6   ATCTGTATCCTTATTTATACCTCTATCTA                                                                      D5S818-R                                                                            (G08446)                                       7, 36                                                                             TGTCATAGTTTAGAACGAACTAAC                                                                           D7S820-F                                                                            106   12 = 90 bp                                                                          GATA                               8   GAAAAACTATCAATCTGTCTATCTAT                                                                         D7S820-R                                                                            (G08616)                                       9, 37                                                                             TTTGTATTTCATGTGTACATTCGTATC                                                                        D8S1179-F                                                                           107   12 = 106 bp                                                                         TATC                               10  ACCTATCCTGTAGATTATTTTCACTGTG                                                                       D8S1179-R                                                                           (G8710)                                        11, 38                                                                            CCCATCTAACGCCTATCTGTATT                                                                            D13S317-F                                                                           108   13 = 122 bp                                                                         TATC                               12  GCCCAAAAAGACAGACAGAAAG                                                                             D13S317-R                                                                           (G09017)                                       13  AGACAGACAGACAGGTGGATAGA                                                                            D16S539-F                                                                           109   11 = 83 bp                                                                          GATA                               14, 39                                                                            TCTCTGTTTTGTCTTTCAATGATA                                                                           D15S539-R                                                                           (G07925)                                       15  TGAGTGACAAATTGAGACCTT                                                                              D18S51-F                                                                            110   13 = 114 bp                                                                         AGAA                               16, 40                                                                            GTCTTACAATAACAGTTGCTACTATT                                                                         D18S51-R                                                                            (L18333)                                       17, 41                                                                            CCCAAGTGAATTGCCTTCTA D21S11-F                                                                            111   26 = 150 bp                                                                         TCTR                               18  GTAGATAGACTGGATAGATAGACGATA                                                                        D21S11-R                                                                            (M84567)                                           G                                                                         19, 42                                                                            GTGTTTTAGATAGATAGATAGGTA                                                                           DYS19-F                                                                             112   10 = 84 bp                                                                          TAGA                               20  GGTTAAGGAGAGTGTCACTA DYS19-R                                                                             (X77751)                                       21, 43                                                                            CAGAGCAAGACTTCATCTG  F13A1-F                                                                             113   7 = 128 bp                                                                          AAAG                               22  TCATTTTAGTGCATGTTC   F13A1-R                                                                             (M21986)                                       23, 44                                                                            TTAGGAGACAAGGATAGCAGTTC                                                                            FES/FPS-F                                                                           114   11 = 91 bp                                                                          ATTT                               24  GCGAAAGAATGAGACTACTATCT                                                                            FES/FPS-R                                                                           (X06292)                                       25, 45                                                                            AAAATTAGGCATATTTACAAGCTAGTT                                                                        FGA-F 115   21 = 142 bp                                                                         CTTT                               26  TCTGTAATTGCCAGCAAAAAAGAAA                                                                          FGA-R (M64982)                                       27, 46                                                                            GTCTCCATCTTTGTCTCTATCTCTATCTG                                                                      HPRTB-F                                                                             116   13 = 108 bp                                                                         TCTA                               28  GAGAAGGGCATGAATTTGCTTT                                                                             HPRTB-R                                                                             (M26434)                                       29  CCTGTTCCTCCCTTATTCCC TH01-F                                                                              117   9 = 79 bp                                                                           TCAT                               30, 47                                                                            GGGAACACAGACTCCATGGT TH01-R                                                                              (D00269)                                       31, 48                                                                            CTTAGGGAACCCTCACTGAATG                                                                             TPOX-F                                                                              118   11 = 89 bp                                                                          AATG                               32  GTCCTTGTCAGCGTTTATTTGC                                                                             TPOX-R                                                                              (M68651)                                       49  GTGAGTTAGCCGTTAGCGAT DYS388-F                                                                            119   17 = 108 bp                                                                         ATT                                50, 83                                                                            GAGCGAGAGTCCGTCTCA   DYS388-R                                                                            (G09695)                                       51, 84                                                                            TTCAATCATACACCCATATCTGTC                                                                           DYS391-F                                                                            120   9 = 99 bp                                                                           TCTR                               52  ATAGAGGGATAGGTAGGCAGGC                                                                             DYS391-R                                                                            G09613                                         53, 85                                                                            TTTTTCTTGTATCACCATT  DYS392-F                                                                            121   16 = 98 bp                                                                          TAT                                54  AAACCTACCAATCCCATTCCTT                                                                             DYS392-R                                                                            G09867                                         55, 85                                                                            TGGTCTTCTACTTGTGTCAATAC                                                                            DYS393-F                                                                            122   15 = 106 bp                                                                         AGAT                               56  TGTCTCATAGAAAAGACATACAT                                                                            DYS393-R                                                                            G09601                                         57, 87                                                                            CTGGATTTCTTGGTTATAGTAAA                                                                            D2S1391-F                                                                           123   12 = 100 bp                                                                         TCTA                               58  AAGCTGGTAGAGAGATACACAGA                                                                            D2S1391-R                                                                           G08168                                         59  AGCCACACCCATAACTTT   D18S535-F                                                                           124   14 = 120 bp                                                                         GATA                               60, 88                                                                            GAATGCAGAGAAAGAGAATCTA                                                                             D18S535-R                                                                           G07985                                         61, 89                                                                            AGAAATGGCTTGGCCTTA   D2S1388-F                                                                           125   11 = 100 bp                                                                         CCTT                               62  TAAAGGATTGCAGGAGGG   D2S1388-R                                                                           G08202                                         63  GAATAAGATTCTGTTGAAGGAAA                                                                            D19S433-F                                                                           126   11 = 100 bp                                                                         AAGG                               64, 90                                                                            AATCTTCTCTCTTTCTACCTCTCT                                                                           D19S433-R                                                                           G08036                                         65, 91                                                                            AGGGCTGATGAGGTGAAATA D6S477-F                                                                            127   16 = 120 bp                                                                         ATCT                               66  TCAACAACAACACATATAAGATGA                                                                           D6S477-R                                                                            G08543                                         67  CATATATTTGTAGATGGATAGAAGA                                                                          D1S518--F                                                                           128   14 = 105 bp                                                                         GATA                               68, 92                                                                            GAGTTCTCCAGAGAAACAGAATC                                                                            D1S518-F                                                                            G07854                                         69, 93                                                                            CAGACTAGATAGATAGATACGTACATA                                                                        D14S306-F                                                                           129   14 = 139 bp                                                                         AGAT                                   CA                                                                        70  TCAAAGAGTGACAAAGAAACTAAA                                                                           D14S306-R                                                                           G09055                                         71, 94                                                                            CCATCCATCTATCATCTATTTATT                                                                           D22S684-F                                                                           130   11 = 100 bp                                                                         TATC                               72  ACCTACATTAGTCTGTGTTCTCT                                                                            D22S684-R                                                                           G08089                                         73, 95                                                                            AAGAAAGAATGACCCTTGGAATTT                                                                           F13B-F                                                                              131   10 = 97 bp                                                                          TTTA                               74  GGGCGACAGAGCAAGACTC  F13B-R                                                                              M64554                                         75, 96                                                                            TGGAGTCGCAAGCTGAACTA CD4-F 132   9 = 108 bp                                                                          TTTTC                              76  CTGAGTGACAGAGTGAGAACCTG                                                                            CD4-R M86525                                         77, 97                                                                            ATCAATGGATGCATAGGTA  D12S391-F                                                                           133   20 = 142 bp                                                                         YAGA                               78  GCCTCCATATCACTTGAGCTAAT                                                                            D13S391-R                                                                           G08921                                         79, 98                                                                            GCCTTACTGACTTACTACATAACGA                                                                          D10S220-F                                                                           134   23 = 100 bp                                                                         CA                                 80  GAGCAAGACTGCATCTCAAAA                                                                              D10S220-R                                                                           Z17087                                         81, 99                                                                            TGGAAAAATATTCTGGGAAGATA                                                                            D7S523-F                                                                            135   17 = 100 bp                                                                         CA                                 66  CCTGTTGACATTTTTAAAACCA                                                                             D7S523-R                                                                            Z17102                                         101 GCCTGTTCCTCCCTTATTTCCC                                                                             TH01-F                                                                              117   9 = bo                                                                              TCAT                               102,                                                                              AGGTCACAGGGAACACAGACTCC                                                                            TH01-R                                                                              D00269                                         103                                                                           __________________________________________________________________________     .sup.1 Bold sequence numbers correspond to primer sequences containing        sequence modifications including biotinylation and the presence of a          cleavable phosphorothiate linkage.                                            .sup.2 F and R indicate forward and reverse primers for each locus.           .sup.3 The sequence listings contain the Genbank sequence for each of the     tandem repeat loci including the DNA tandem repeat region and flanking        regions for each locus. The sequence listings correspond to only a portio     of the full Genbank sequence listing.                                         .sup.4 The first number in the PCR product size is the number of repeats      found in the Genbank sequence listing for each locus and the second is th     predicted size of PCR product from the Genbank sequence when using the        listed primers to amplify the tandem repeat locus. Of course, the number      of tandem repeats within a population of individuals will vary and            therefore so will the PCR product size when individual samples are            analyzed.                                                                     .sup.5 Repeats sequence nomenclature used here is according to the latest     recommendations of the DNA Commission of the International Society for        Forensic Haemogenetics, and described in Int. J. Legal Med. 110:175-176       (1997).                                                                  

At least one of the primers in each locus-specific pair contains abiotin moiety at the 5'-end and a phosphorothioate linkage attached to aT near the 3'-end for a capture and release assay usingstreptavidin-coated magnetic beads (PCT Patent Application No. WO96/37630). Although many of the specific primers of the presentinvention are designed for use in such a capture and release assay, suchprimers need not contain either solid-binding or cleavable sites, or maycontain any combination of them.

The purpose of such an assay is to increase mass resolution by (1)allowing for higher purities of the nucleic acid extension product and(2) decreasing the size of the nucleic acid extension product. Bindingto a solid support fulfills the first goal by allowing for stringentwashes and removing the complementary strand of the DNA which providescumulative information and complicates the mass spectra decreasing theresolution.

This assay may be used to isolate single-stranded or double-strandedamplified target nucleic acids. Typically, at least one strand of anamplified target nucleic acid is bound to a solid support to permitrigorous washing and concomitant removal of salt adducts, unwantedoligonucleotides and enzymes. Either a double-stranded amplified targetnucleic acid or a single-stranded amplified target nucleic acid may beisolated for mass spectrometric analysis depending upon the stringencyof the wash. The single-stranded amplified target nucleic acid analyzedmay be either the strand bound or not bound to the solid support. If theunbound strand is used for MS analysis, it is purified by first washingthe bound strand and its attached complement under conditions notsufficiently rigorous to disrupt the strand. After unwanted biomoleculesand salts are removed, the complement can then be released under morerigorous conditions. Cleavable linkers or cleavable primers may then beused to release the bound strands from the solid support prior to MSanalysis.

The second goal is met by having cleavable sites in the primer. Suchcleavable sites also eliminate unnecessary sequences and allow for theuse of a capture and release assay and for primer modification for theinterleaving multiplexing method, described herein. For example, movingthe cleavable site along the primer backbone directly modifies the massof the PCR™ product. The cleavable site is typically introduced via acleavable primer and the cleavable site is located outside of the regionof interest. Cleavable primers may include those comprising anexonuclease blocking moiety, a Type IIS restriction endonucleaserecognition site, and a Type II restriction endonuclease recognitionsite.

The target nucleic acids may, thus, be reduced in length by any of themethods known that will cleave within one or more flanking regionspreferably without cleaving within the region of interest. Exemplarymethods of reducing length include: cleaving at endogenous restrictionendonuclease cleavable sites present in one or more flanking regions butabsent in the region of interest; cleaving at restriction endonucleasecleavable sites at or adjacent to restriction endonuclease recognitionsites incorporated into one or more flanking regions by use of one ormore cleavable primers comprising said restriction endonucleaserecognition sites; cleaving at a combination of restriction endonucleasecleavable sites wherein the sites are endogenous and/or introduced usingmismatch or overhanging primers; and selective digestion of one or moreflanking regions using exonuclease and an exonuclease blocking moiety toprotect the regions of interest from digestion.

The restriction endonucleases employed with the present inventioninclude type II and type IIS restriction endonucleases. The restrictionendonuclease recognition sites may be either within a primer region, oroutside the primer region, so long as the restriction endonucleasecleavable sites are within one or more flanking regions and preferablynot within a region of interest. For type II restriction endonucleases,the restriction endonuclease recognition site is the same as therestriction endonuclease cleavable site. For Type IIS restrictionendonucleases, the cleavable site is at a defined distance away from oneside of the recognition site.

Another embodiment of the invention involves using a cleavable primerhaving an exonuclease blocking moiety. After amplification of the targetnucleic acid, the amplified target nucleic acid will include anexonuclease blocking moiety. The amplified target nucleic acid is thentreated with a 5' to 3' exonuclease, which degrades the strandcontaining the exonuclease blocking moiety in a 5' to 3' direction onlyup to the blocking moiety. The 5' to 3' exonuclease may optionallydegrade the other complementary strand of the amplified target nucleicacid, in cases where the other strand does not have an exonucleaseblocking moiety. The treatment with the 5' to 3' exonuclease leaves areduced-length, single-stranded amplified target nucleic acid for massspectrometric analysis.

Cleavable sites may also include chemically cleavable groupsincorporated within the phosphate backbone linkage (e.g replacement ofphosphate with a phosphoramidate) or as a substituent on or replacementof one of the bases or sugars of the oligonucleotide primer (e.g. amodified base or sugar, for example, a more labile glycosidic linkage).Such chemically cleavable groups would be apparent to one of skill inthe art in light of the present disclosure and include, for example,dialkoxysilane, 3'-(S)-phosphorothioate, 5'-(S)-phosphorothioate,3'-(N)-phosphoroamidate, 5'-(N)-phosphoroamidate, and ribose. FIGS. 16Aand 16B depict a 3'-(S)-phosphorothioate and 5'-(S)-phosphorothioate,respectively as defined in this invention. Note that these linkages areoften referred to as thiophosphates as well. A similar nomenclature isemployed for 3'-(N)-phosphoroamidate, 5'-(N)-phosphoroamidate. Thechemically cleavable site should generally be stable under theamplification, hybridization and washing conditions to be employed andis preferably within one or more of the flanking regions.

In a preferred embodiment, the cleavable site is located near the 3' endof the primer used to bind the amplified target nucleic acid to thesolid support. By locating the cleavable site near the 3' end, it ispossible to further reduce the length of the amplified target nucleicacid, eliminating a flanking region from the polynucleotide region ofinterest. Cleavable primers are described in PCT/US96/06116, filed Apr.26, 1996 (incorporated herein by reference).

The primer pairs described in this invention may be combined to generateoverlapping PCR™ product sizes which are all distinguishable by mass.

EXAMPLE 1 PCR CONDITIONS FOR MULTIPLEXING DTNR RESULTS

Template: 5 uL 1:1000 dilution of AmpFISTR Green I Allelic Ladders (PEApplied Biosystems; contains common alleles from the STR loci CSF1PO,TPOX, and TH01 and the sex-typing marker amelogenin); for regularsamples, 2-5 uL of 1-10 ng of human genomic DNA was added to the PCRreaction.

Reaction Mix: 20 uL reaction with 1× STR buffer (Promega; contains 1.5mM MgCl₂, 200 uM dNTPs, etc.), 1 U Taq polymerase (Promega), 20 pmolforward and reverse primers with one of them being a primer containing abiotin moiety on the 5'-end and a thiothymine residue near the 3'-end ofthe oligonucleotide.

Thermal Cycling: In 0.2 mL tubes in an MJ Research DNA Engine (blocktemperature) 94° C. for 2 min; 35 cycles: 94° C. for 30 sec, 60° C. for30 sec, 72° C. for 30 sec; 72° C. for 5 min.

EXAMPLE 2 SAMPLE PURIFICATION FOR MULTIPLEXING DTNR RESULTS

A typical binding/washing protocol for purifying samples for DTNRmultiplexing includes the following steps:

a) Wash 10 uL streptavidin-coated magnetic beads with 2× binding/washbuffer

b) Repeat a second time

c) Add 5 uL 5× binding/wash buffer then add ˜19 uL of PCR sample to thebeads (1 uL was removed for an agarose gel check) and vortex sample tubefor 15 min at slow speed

d) Wash beads with 30 uL of 2× binding/wash buffer

e) Wash beads with 30 uL of 0.1 N NaOH

f) Add 30 uL of 0.1 N NaOH and vortex for 10 min at slow speed

g) Wash beads with 30 uL of 0.1 N NaOH

h) Wash beads with 30 uL of 20 mM ammonium acetate

i) Repeat step (h) five times

j) Wash beads with deionized water

k) Repeat step (j) twice

Note after each step, the supernatant is removed while the beads aremagnetically held in the bottom of the tube.

After purification the solid bound strands were released by cleaving atthe chemically cleavable thiophosphate site by the following procedure:7 uL of 0.1 mM silver nitrate was added and the samples were incubatedat 48° C. for 15 min.; the supernatant was then transferred to a cleantube and 2 uL of 70 mM DTT was added; and finally the sample was driedin a speed vacuum. For mixed samples the preceding protocol was modifiedin that aliquots of the samples (e.g., 3 uL TH01 ladder+3 uL TPOXladder) were mixed before the drying step.

EXAMPLE 3 MS ANALYSIS FOR MULTIPLEXING DTNR RESULTS

The matrix consisted of a 5:1 molar ratio of 3-hydroxypicolinic acid(3-HPA; Lancaster Synthesis) to picolinic acid (PA; Aldrich) and wasprepared by mixing 18 uL of a freshly prepared saturated 3-HPA solution(˜0.5 M) with 2 uL of 1 M PA

The sample to be analyzed was reconstituted in 0.5 uL of the matrix andmanually spotted on the sample plate.

The instrument conditions employed with a linear time-of-flight massspectrometer consisted of the following: acceleration voltage of +20 kV;delay of +3.6 kV at 1.12 usec; laser setting of 179 on the polarizer;mass gate of 5.84 usec; and 400 shots. A 2-point mass calibration with a15-mer (4507.0 Da) and a 36-mer (10998.2 Da) was employed.

EXAMPLE 4

Oligonucleotide primers are typically prepared by the phosphoramiditeapproach. In this automated, solid-phase procedure, each nucleotide isindividually added to the 5'-end of the growing oligonucleotide chain,which is in turn attached at the 3'-end to a solid support. The addednucleotides are in the form of trivalent 3'-phosphoramidites that areprotected from polymerization by a dimethoxytrityl ("DMT") group at the5'-position. After base induced phosphoramidite coupling, mild oxidationto give a pentavalent phosphotriester intermediate and DMT removalprovides a new site for oligonucleotide elongation. These syntheses maybe performed on a Perkin Elmer/Applied Biosystems Division DNAsynthesizer. The oligonucleotide primers are then cleaved off the solidsupport, and the phosphodiester and exocyclic amino groups aredeprotected with ammonium hydroxide.

The biotin, and 3'- and 5'-(S) phosphorothioate linkages are alsoprepared in an automated fashion from phosphoramidite intermediatesusing similar procedures and either modified bases or activated andprotected linker molecules.

EXAMPLE 5 TWO STAGE MULTIPLEXING REACTION OUTSIDE PRIMERS FOR HIGHERLEVEL MULTIPLEX FOLLOWED BY SINGLE DDN TERMINATION

A triplex PCR reaction was run with 10-ng human genomic DNA template ina 20-uL PCR reaction. Primers specific for the three STR loci CSF1PO,TPOX, and TH01 were used as described by Huang et al. These primersproduce larger sized PCR products than the primers listed in this patentand the primer sequences from Table 1 for these three STR loci arewithin the product region.

Multiplex PCR components: 20 μL reaction containing 1.5× Taq buffer II(PE Applied Biosystems), 200 μM dNTPs, 1.5 mM MgCl₂, 1 U AmpliTaq Gold(PE Applied Biosystems), 0.5 μM each primer.

Thermal cycling was performed in 0.2 mL tubes using an MJ Research DNAEngine (calculated temperature) with the following cycling parameters:95° C. for 11 min; 40 cycles: 94° C. for 30 sec, 64° C. for 30 sec, 68°C. for 45 sec; 70° C. for 10 min.

Following PCR, the sample was treated with shrimp-alkaline phosphatase(SAP) to hydrolyze the unincorporated dNTPs. Typically, 1 U SAP wasadded to each 20 μL PCR sample. The sample was then incubated at 37° C.for 60 minutes followed by heating at 75° C. for 15 minutes.

A multiplexed primer extension assay was then performed using cleavableprimers for the three STR loci. The reaction included three regulardeoxynucleotide triphosphates (dNTP) to allow incorporation through theSTR repeat region and a single dideoxynucleotide triphosphate (ddNTP) tohalt extension (see Braun, et al.). The termination by the ddNTPshortened the measured STR allele.

Multiplexed primer extension reaction components: 20 μL reactioncontaining 1× TaqFS buffer (PE Applied Biosystems), 2.4 U TaqFS, 200 μMdGTP, 200 μM dATP, 200 μM dTTP, 400 μM ddCTP, 40 pmol CSF1PO primer (SEQID NO:100), 20 pmol TPOX (SEQ ID NO:31), 20 pmol TH01 (SEQ ID NO:103),and 2 μL SAP-treated PCR product (as described above).

Thermal cycling for extension reaction was performed in 0.2 mL tubesusing an MJ Research DNA Engine (calculated temperature) with thefollowing cycling parameters: 95° C. for 2 min; 30 cycles: 94° C. for 30sec, 55° C. for 30 sec, 72° C. for 45 sec; 60° C. for 5 min. Theresultant product was purified and analyzed as detailed in the examplesabove.

As seen in FIG. 8A, the possible alleles including microvariants, suchas TH01 9.3, are resolvable from one another in all three STR systemseven though they overlap in the mass range. FIG. 8B illustrates a resultwith this particular STR multiplex. The sample contains a homozygousTPOX allele 8, heterozygous TH01 alleles 6 and 9.3, and a homozygousCSF1PO allele 12. In this particular case, the strand containing an AATGor ATAG repeat was used in all three STR loci so that the samedideoxynucleotide triphoshate (ddC) could be used to terminate thestrand on the opposite side of the repeat from the cleavable primer.After the extension reaction with the ddNTP and the cleavage reaction,the repeat region and only a flanking few bases on either side of therepeat are present for the three STR loci. Mass accuracy as well asresolution and sensitivity are improved in the mass spectrometer bygoing to smaller sizes for the STR alleles using this approach.

EXAMPLE 6 TWO STAGE MULTIPLEXING REACTION OUTSIDE PRIMERS FOR HIGHERLEVEL MULTIPLEX FOLLOWED BY GTS PRIMERS IN LOWER LEVEL MULTIPLEX THATPRODUCE SMALLER PCR PRODUCTS

In a situation where there is a small or limited amount of DNA templateavailable, a highly multiplexed PCR reaction may be performed initiallyfollowed by a second stage PCR with primers that are interior (i.e.,closer to the repeat region) than those contained in the first multiplex("nested PCR"). The first multiplex could include, for example, 8-14 STRloci that are PCR-amplified simultaneously. Aliquots of these ampliconscould then be divided and reamplified in a second PCR reaction withprimers for a subset of the STR loci already amplified. For example, sixduplex reactions or four triplexes with mass spectroscopy primers couldbe performed to measure all of the STR loci in an initial 12-plexreaction. Primers used in these duplexes could be from those listed inTable 1. Provided that the primers for the first stage multiplex areoutside (or at least identical to) the second stage primer sets, thisapproach will work for any PCR-compatible primers.

The advantage of the nested PCR approach is that a SAP-treatment is notrequired (as in Example 5) before the second stage reaction as dNTPs areused. However, measured STR alleles will be longer and thus morechallenging to analyze in the mass spectrometer than STR allele productscreated with the primer termination reaction (ddN) approach describedabove.

EXAMPLE 7 FTA PAPER USED IN PCR REACTIONS IN PLACE OF EXTRACTED DNA

Liquid blood was stained on an FTA™ Card (Life Technologies,Gaithersburg, Md.) and allowed to air-dry for 1 hour. A small portion ofblood-stained paper was cut out of the center of the spot and placed ina 0.6 mL tube. An aliquot of 50 μL FTA™ Purification Reagent (LifeTechnologies) was added to the tube and vortexed for several seconds.The tube was allowed to sit for 5 minutes at room temperature. The tubewas vortexed again and the liquid in the tube was removed. Anotheraliquot of 50 μL FTA™ Purification Reagent was added to the tube and thevortexing and waiting steps were repeated as described above. The FTA™paper was then washed a third time with FTA™ Purification Reagent andthen washed twice more with deionized water. After these washes, theliquid was removed with a pipet and the FTA™ paper punch was left in thetube to air-dry for 1 hour.

The dried punch was added directly to PCR amplification mix in place ofextracted human genomic DNA. PCR was performed as described in the otherexamples with no changes to amplification conditions or reagents. Nodecrease in PCR efficiency was observed when the FTA™ paper was comparedto standard K562 human genomic DNA templates. The use of FTA™ papergreatly facilitates the automation of DNA typing procedures as extensiveDNA extraction is not needed from liquid blood samples.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

U.S. Pat. No. 4,683,202 Mullis

U.S. Pat. No. 5,364,759 Caskey et al.

U.S. Pat. No. 5,378,602 Polymeropoulos et al.

U.S. Pat. No. 5,599,666 Schumm et al.

U.S. Pat. No. 5,605,798 Koster

U.S. Pat. No. 5,700,642 Monforte et al.

U.S. Pat. No. 5,674,686 Schumm and Puers

U.S. Pat. No. 5,766,847 Jackie and Tautz

U.S. Pat. No. 5,496,562 Burgoyne

Alford, Hammond, Coto, Caskey, "Rapid and efficient resolution ofparentage by amplification of short tandem repeats," Am. J. Hum. Genet.,55: 190-195, 1994.

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Becker, Li, Shaler, Hunter, Lin, Monforte, "Genetic analysis of shorttandem repeat loci by time of flight mass spectrometry," SeventhInternational Symposium on Human Identification (1996), pp. 158-162,1997.

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Fregeau and Fourney, "DNA typing with fluorescently tagged short tandemrepeats: a sensitive and accurate approach to human identification,"BioTechniques, 15:100-119, 1993.

Hammond, Jin, Zhong, Caskey, Chakraborty, "Evaluation of 13 short tandemrepeat loci for use in personal identification applications," Am. J.Hum. Genet., 55:175-189, 1994.

Hauge and Litt, "A study of the origin of `shadow bands` seen whentyping dinucleotide repeat polymorphisms by the PCR™," Hum. Mol. Genet.,2:411-415, 1993.

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Kimpton, Walton, Gill, "A further tetranucleotide repeat polymorphism inthe vWF gene," Hum. Mol. Genet., 1:287, 1992.

Kimpton, Gill, Walton, Urquhart, Millican, Adams, "Automated DNAprofiling employing multiplex amplification of short tandem repeatloci," PCR™ Meth. Appl., 3:13-22, 1993.

Kimpton, Oldroyd, Watson, Frazier, Johnson, Millican, Urquhart, Sparkes,Gill, "Validation of highly discriminating multiplex short tandem repeatamplification systems for individual identification," Electrophoresis,17:1283-1293, 1996.

Lareu, Pestoni, Schurenkamp, Rand, Brinkmann, Carracedo, "A highlyvariable STR at the D12S391 locus," Int. J. Leg. Med., 109:134-138,1996.

Lygo, Johnson, Holdaway, Woodroffe, Whitaker, Clayton, Kimpton, Gill,"The validation of short tandem repeat (STR) loci for use in forensiccasework," Int. J. Leg. Med., 107:77-89, 1994.

Polymeropoulos, Rath, Xiao, Merril, "Tetranucleotide repeat polymorphismat the human c-fes/fps proto-oncogene (FES)," Nucleic Acids Res.,19:4018, 1991.

Polymeropoulos, Rath, Xiao, Merril, "Tetranucleotide repeat polymorphismat the human coagulation factor XIII A subunit gene (F13A1)," NucleicAcids Res., 19:4306, 1991.

Polymeropoulos, Xiao, Rath, Merril, "Tetranucleotide repeat polymorphismat the human tyrosine hydroxylase gene (TH)," Nucleic Acids Res.,19:3753, 1991.

Puers, Hammond, Caskey, Lins, Sprecher, Brinkmann, Schumm, "Alleleladder characterization of the short tandem repeat polymorphism locatedin the 5' flanking region to the human coagulation factor XIII A subunitgene," Genomics, 23:260-264, 1994.

Puers, Hammond, Jin, Caskey, Schumm, "Identification of repeat sequenceheterogeneity at the polymorphic short tandem repeat locusHUMTH01[AATG]n and reassignment of alleles in population analysis byusing a locus-specific allele ladder," Am. J. Hum. Genet., 53:953-958,1993.

Roewer, Arnemann, Spurr, Grzeschik, Epplen, "Simple repeat sequences onthe human Y chromosome are equally polymorphic as their autosomalcounterparts," Hum. Genet., 89:389-394, 1992.

The Utah Marker Development Group "A collection of orderedtetranucleotide-repeat markers from the human genome," Am. J. Hum.Genet., 57:619-628, 1995.

Weber and May, "Abundant class of human DNA polymorphisms which can betyped using the polymerase chain reaction," Am. J. Hum. Genet.,44:388-396, 1989.

Ziegle, Su, Corcoran, Nie, Mayrand, Hoff, McBride, Kronick, Diehl,"Application of automated DNA sizing technology for genotypingmicrosatellite loci," Genomics, 14:1026-1031,1992.

Braun, A., et al., "Detecting CFTR gene mutations by using primer oligobase extension and mass spectrometry," Clin. Chem. 43:1151-1158 (1997).

Braun, A., et al., "Improved Analysis of Microsatellites Using MassSpectrometry," Genomics 46:18-23 (1997).

Butler, J. M., et al., "Reliable Genotyping of Short Tandem Repeat Lociwithout an Allelic Ladder Using Time-of-Flight Mass Spectrometry," Int.J. Legal Med., in press (1998).

Butler, J. M., et al., "Rapid and Automated Analysis of Short TandemRepeat Loci Using Time-of-Flight Mass Spectrometry," Proceedings of theEighth International Symposium on Human Identification 1997, PromegaCorporation, pp. 94-101 (1998).

Butler, J. M., et al., "High-throughput STR Analysis by Time-of-FlightMass Spectrometry," Proceedings of the Second European Symposium onHuman Identification 1998, Promega Corporation, in press (1998).

Huang, N. E., et al., "Chinese population data on three tetrameric shorttandem repeat loci-HUMTH01, TPOX, and CSF1PO-derived using multiplex PCRand manual typing," Forensic Sci. Int. 71:131-136 (1995).

Kayser, M., et al., "Evaluation of Y-chromosomal STRs: a multicenterstudy," Int. J. Legal Med. 110:125-133 (1997).

Little, D. P., et al., "MALDI on a Chip: Analysis of Arrays ofLow-Femtomole to Subfemtomole Quantities of Synthetic Oligonucleotidesand DNA Diagnostic Products Dispensed by a Piezoelectric Pipet," Anal.Chem. 69:4540-4546 (1997).

Little, D. P., et al., "Mass Spectrometry from Miniaturized Arrays forFull Comparative DNA Analysis," Nature Med. 3:1413-1416 (1997).

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Ross, P. L., et al., "Analysis of DNA Fragments from Conventional andMicrofabricated PCR Devices Using Delayed Extraction MALDI-TOF MassSpectrometry," Anal. Chem. 70:2067-2073 (1998).

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                - <160> NUMBER OF SEQ ID NOS: 135                                             - <210> SEQ ID NO 1                                                           <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 1                                                           #                23taga tag                                                   - <210> SEQ ID NO 2                                                           <211> LENGTH: 22                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (21)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 2                                                           #                 22aag ta                                                    - <210> SEQ ID NO 3                                                           <211> LENGTH: 19                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 3                                                           # 19               ggt                                                        - <210> SEQ ID NO 4                                                           <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (19)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 4                                                           # 20               cttg                                                       - <210> SEQ ID NO 5                                                           <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (24)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 5                                                           #               25 tgta atatt                                                 - <210> SEQ ID NO 6                                                           <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 6                                                           #            29    atac ctctatcta                                             - <210> SEQ ID NO 7                                                           <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (21)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-phospho - #rothioate            - <400> SEQUENCE: 7                                                           #                24gaac taac                                                  - <210> SEQ ID NO 8                                                           <211> LENGTH: 26                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 8                                                           #              26  tcta tctatc                                                - <210> SEQ ID NO 9                                                           <211> LENGTH: 27                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (26)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 9                                                           #             27   acat tcgtatc                                               - <210> SEQ ID NO 10                                                          <211> LENGTH: 28                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 10                                                          #             28   attt tcactgtg                                              - <210> SEQ ID NO 11                                                          <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (22)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 11                                                          #                23ctgt att                                                   - <210> SEQ ID NO 12                                                          <211> LENGTH: 22                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 12                                                          #                 22gaa ag                                                    - <210> SEQ ID NO 13                                                          <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 13                                                          #                23ggat aga                                                   - <210> SEQ ID NO 14                                                          <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (23)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 14                                                          #                24caat gata                                                  - <210> SEQ ID NO 15                                                          <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 15                                                          #21                acct t                                                     - <210> SEQ ID NO 16                                                          <211> LENGTH: 26                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (25)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 16                                                          #              26  tgct actatt                                                - <210> SEQ ID NO 17                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (19)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 17                                                          # 20               tcta                                                       - <210> SEQ ID NO 18                                                          <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 18                                                          #            29    gata gacgataga                                             - <210> SEQ ID NO 19                                                          <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (23)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 19                                                          #                24gata ggta                                                  - <210> SEQ ID NO 20                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 20                                                          # 20               acta                                                       - <210> SEQ ID NO 21                                                          <211> LENGTH: 19                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (16)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 21                                                          # 19               ctg                                                        - <210> SEQ ID NO 22                                                          <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 22                                                          #  18              tc                                                         - <210> SEQ ID NO 23                                                          <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (22)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 23                                                          #                23gcag ttc                                                   - <210> SEQ ID NO 24                                                          <211> LENGTH: 22                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 24                                                          #                 22cat ct                                                    - <210> SEQ ID NO 25                                                          <211> LENGTH: 27                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (26)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 25                                                          #             27   acaa gctagtt                                               - <210> SEQ ID NO 26                                                          <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 26                                                          #               25 aaaa agaaa                                                 - <210> SEQ ID NO 27                                                          <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (28)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 27                                                          #            29    ctat ctctatctg                                             - <210> SEQ ID NO 28                                                          <211> LENGTH: 22                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 28                                                          #                 22gct tt                                                    - <210> SEQ ID NO 29                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 29                                                          # 20               tccc                                                       - <210> SEQ ID NO 30                                                          <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (20)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 30                                                          #21                tggt g                                                     - <210> SEQ ID NO 31                                                          <211> LENGTH: 22                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (21)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 31                                                          #                 22gaa tg                                                    - <210> SEQ ID NO 32                                                          <211> LENGTH: 22                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 32                                                          #                 22ttt gc                                                    - <210> SEQ ID NO 33                                                          <211> LENGTH: 22                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 33                                                          #                 22aag ta                                                    - <210> SEQ ID NO 34                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 34                                                          # 20               cttg                                                       - <210> SEQ ID NO 35                                                          <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 35                                                          #               25 tgta atatt                                                 - <210> SEQ ID NO 36                                                          <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 36                                                          #                24gaac taac                                                  - <210> SEQ ID NO 37                                                          <211> LENGTH: 27                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 37                                                          #             27   acat tcgtatc                                               - <210> SEQ ID NO 38                                                          <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 38                                                          #                23ctgt att                                                   - <210> SEQ ID NO 39                                                          <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 39                                                          #                24caat gata                                                  - <210> SEQ ID NO 40                                                          <211> LENGTH: 26                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 40                                                          #              26  tgct actatt                                                - <210> SEQ ID NO 41                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 41                                                          # 20               tcta                                                       - <210> SEQ ID NO 42                                                          <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 42                                                          #                24gata ggta                                                  - <210> SEQ ID NO 43                                                          <211> LENGTH: 19                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 43                                                          # 19               ctg                                                        - <210> SEQ ID NO 44                                                          <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 44                                                          #                23gcag ttc                                                   - <210> SEQ ID NO 45                                                          <211> LENGTH: 27                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 45                                                          #             27   acaa gctagtt                                               - <210> SEQ ID NO 46                                                          <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 46                                                          #            29    ctat ctctatctg                                             - <210> SEQ ID NO 47                                                          <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 47                                                          #21                tggt g                                                     - <210> SEQ ID NO 48                                                          <211> LENGTH: 22                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 48                                                          #                 22gaa tg                                                    - <210> SEQ ID NO 49                                                          <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 49                                                          #21                gcga t                                                     - <210> SEQ ID NO 50                                                          <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 50                                                          #  18              ca                                                         - <210> SEQ ID NO 51                                                          <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 51                                                          #                24tatc tgtc                                                  - <210> SEQ ID NO 52                                                          <211> LENGTH: 22                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 52                                                          #                 22cag gc                                                    - <210> SEQ ID NO 53                                                          <211> LENGTH: 19                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 53                                                          # 19               att                                                        - <210> SEQ ID NO 54                                                          <211> LENGTH: 22                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 54                                                          #                 22tcc tt                                                    - <210> SEQ ID NO 55                                                          <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 55                                                          #                23tcaa tac                                                   - <210> SEQ ID NO 56                                                          <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 56                                                          #                23cata cat                                                   - <210> SEQ ID NO 57                                                          <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 57                                                          #                23tagt aaa                                                   - <210> SEQ ID NO 58                                                          <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 58                                                          #                23acac aga                                                   - <210> SEQ ID NO 59                                                          <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 59                                                          #  18              tt                                                         - <210> SEQ ID NO 60                                                          <211> LENGTH: 22                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 60                                                          #                 22atc ta                                                    - <210> SEQ ID NO 61                                                          <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 61                                                          #  18              tg                                                         - <210> SEQ ID NO 62                                                          <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 62                                                          #  18              gg                                                         - <210> SEQ ID NO 63                                                          <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 63                                                          #                23aagg aaa                                                   - <210> SEQ ID NO 64                                                          <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 64                                                          #                24acct ctct                                                  - <210> SEQ ID NO 65                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 65                                                          # 20               aata                                                       - <210> SEQ ID NO 66                                                          <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 66                                                          #                24taag atga                                                  - <210> SEQ ID NO 67                                                          <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 67                                                          #               25 gata gaaga                                                 - <210> SEQ ID NO 68                                                          <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 68                                                          #                23caga atc                                                   - <210> SEQ ID NO 69                                                          <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 69                                                          #            29    atac gtacataca                                             - <210> SEQ ID NO 70                                                          <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 70                                                          #                24aaac taaa                                                  - <210> SEQ ID NO 71                                                          <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 71                                                          #                24tatt tatt                                                  - <210> SEQ ID NO 72                                                          <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 72                                                          #                23gttc tct                                                   - <210> SEQ ID NO 73                                                          <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 73                                                          #                24tgga attt                                                  - <210> SEQ ID NO 74                                                          <211> LENGTH: 19                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 74                                                          # 19               ctc                                                        - <210> SEQ ID NO 75                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 75                                                          # 20               acta                                                       - <210> SEQ ID NO 76                                                          <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 76                                                          #                23gaac ctg                                                   - <210> SEQ ID NO 77                                                          <211> LENGTH: 19                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 77                                                          # 19               gta                                                        - <210> SEQ ID NO 78                                                          <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 78                                                          #                23agct aat                                                   - <210> SEQ ID NO 79                                                          <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 79                                                          #               25 acat aacga                                                 - <210> SEQ ID NO 80                                                          <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 80                                                          #21                caaa a                                                     - <210> SEQ ID NO 81                                                          <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 81                                                          #                23gaag ata                                                   - <210> SEQ ID NO 82                                                          <211> LENGTH: 22                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 82                                                          #                 22aac ca                                                    - <210> SEQ ID NO 83                                                          <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (16)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 83                                                          #  18              ca                                                         - <210> SEQ ID NO 84                                                          <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (23)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 84                                                          #                24tatc tgtc                                                  - <210> SEQ ID NO 85                                                          <211> LENGTH: 19                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (18)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 85                                                          # 19               att                                                        - <210> SEQ ID NO 86                                                          <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (21)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 86                                                          #                23tcaa tac                                                   - <210> SEQ ID NO 87                                                          <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (20)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 87                                                          #                23tagt aaa                                                   - <210> SEQ ID NO 88                                                          <211> LENGTH: 22                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (21)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 88                                                          #                 22atc ta                                                    - <210> SEQ ID NO 89                                                          <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (17)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 89                                                          #  18              tg                                                         - <210> SEQ ID NO 90                                                          <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (22)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 90                                                          #                24acct ctct                                                  - <210> SEQ ID NO 91                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (19)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 91                                                          # 20               aata                                                       - <210> SEQ ID NO 92                                                          <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (22)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 92                                                          #                23caga atc                                                   - <210> SEQ ID NO 93                                                          <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (26)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 93                                                          #            29    atac gtacataca                                             - <210> SEQ ID NO 94                                                          <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (23)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 94                                                          #                24tatt tatt                                                  - <210> SEQ ID NO 95                                                          <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (23)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 95                                                          #                24tgga attt                                                  - <210> SEQ ID NO 96                                                          <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (19)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 96                                                          # 20               acta                                                       - <210> SEQ ID NO 97                                                          <211> LENGTH: 19                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (18)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 97                                                          # 19               gta                                                        - <210> SEQ ID NO 98                                                          <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (20)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 98                                                          #               25 acat aacga                                                 - <210> SEQ ID NO 99                                                          <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (22)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 99                                                          #                23gaag ata                                                   - <210> SEQ ID NO 100                                                         <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (21)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 100                                                         #                23taga tag                                                   - <210> SEQ ID NO 101                                                         <211> LENGTH: 22                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 101                                                         #                 22ttc cc                                                    - <210> SEQ ID NO 102                                                         <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 102                                                         #                23agac tcc                                                   - <210> SEQ ID NO 103                                                         <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: Biotinylated                                         <220> FEATURE:                                                                <221> NAME/KEY: misc.sub.-- feature                                           <222> LOCATION: (21)                                                          <223> OTHER INFORMATION: 2'-deoxythymidine-5'-(S)-pho - #sphorothioate        - <400> SEQUENCE: 103                                                         #                23agac tcc                                                   - <210> SEQ ID NO 104                                                         <211> LENGTH: 315                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 104                                                         - aacctgagtc tgccaaggac tagcaggttg ctaaccaccc tgtgtctcag tt - #ttcctacc         60                                                                          - tgtaaaatga agatattaac agtaactgcc ttcatagata gaagatagat ag - #attagata        120                                                                          - gatagataga tagatagata gatagataga tagatagata gataggaagt ac - #ttagaaca        180                                                                          - gggtctgaca caggaaatgc tgtccaagtg tgcaccagga gatagtatct ga - #gaaggctc        240                                                                          - agtctggcac catgtgggtt gggtgggaac ctggaggctg gagaatgggc tg - #aagatggc        300                                                                          #   315                                                                       - <210> SEQ ID NO 105                                                         <211> LENGTH: 307                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 105                                                         - tctaattaaa gtggtgtccc agataatctg tactaataaa agtatatttt aa - #tagcaagt         60                                                                          - atgtgacaag ggtgattttc ctctttggta tccttatgta atattttgaa ga - #tagataga        120                                                                          - tagatagata gatagataga tagatagata gataggtaga tagaggtata aa - #taaggata        180                                                                          - cagatatagn tacaaatgtt gtaaactgtg gctatgattg gaatcacttg gc - #taaaaagc        240                                                                          - gctnaagcnt tcctctgnga gaggcaatta cttttttnct taggnactnc ct - #cancagtc        300                                                                          #         307                                                                 - <210> SEQ ID NO 106                                                         <211> LENGTH: 334                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 106                                                         - aatttttgta ttttttttag agacggggtt tcaccatgtt ggtcaggctg ac - #tatggagt         60                                                                          - tattttaagg ttaatatata taaagggtat gatagaacac ttgtcatagt tt - #agaacgaa        120                                                                          - ctaacgatag atagatagat agatagatag atagatagat agatagatag at - #agacagat        180                                                                          - tgatagtttt tttttatctc actaaatagt ctatagtaaa catttaatta cc - #aatatttg        240                                                                          - gtgcaattct gtcaatgagg ataaatgtgg aatcgttata attcttaaga at - #atatattc        300                                                                          #       334        acct cagattttaa ggcc                                       - <210> SEQ ID NO 107                                                         <211> LENGTH: 340                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 107                                                         - tggcaactta tatgtatttt tgtatttcat gtgtacattc gtatctatct at - #ctatctat         60                                                                          - ctatctatct atctatctat ctatctatct attccccaca gtgaaaataa tc - #tacaggat        120                                                                          - aggtaaataa attaaggcat attcacgcaa tgggatacgn tacagtgatg aa - #aatgaact        180                                                                          - aattatagct acgtgaaact atactcatgn acacaatttg gtaaaagaaa ct - #gggaacaa        240                                                                          - gaatacatac ggtttttgnc agctgtgcta ttttacattc ccaacaacaa tg - #cacagggt        300                                                                          #   340            nctt gtcaacattn tgttattttg                                 - <210> SEQ ID NO 108                                                         <211> LENGTH: 286                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 108                                                         - tgggatgggt tgctggacat ggtatcacag aagtctggga tgtggaggag ag - #ttcatttc         60                                                                          - tttagtgggc atccgtgact ctctggactc tgacccatct aacgcctatc tg - #tatttaca        120                                                                          - aatacattat ctatctatct atctatctat ctatctatct atctatctat ct - #atctatca        180                                                                          - atcatctatc tatctttctg tctgtctttt tgggctgcct atggctcaac cc - #aagttgaa        240                                                                          #                286caa ttcaagctct ctgaatatgt tttgaa                          - <210> SEQ ID NO 109                                                         <211> LENGTH: 426                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 109                                                         - atggctgccc tcacggctgc accgggagga tgactgtntt cccactctca gt - #cctgccga         60                                                                          - ggtgcctgac agccctgcac ccaggagctg gggggtctaa gagcttgtaa aa - #agtgtaca        120                                                                          - agtgccagat gctcgttgtg cacaaatcta aatgcagaaa agcactgaaa ga - #agaatcca        180                                                                          - gaaaaccaca gttcccattt ttatatggga gcaaacaaag gcagatccca ag - #ctcttcct        240                                                                          - cttccctaga tcaatacaga cagacagaca ggtggataga tagatagata ga - #tagataga        300                                                                          - tagatagata gatagatatc attgaaagac aaaacagaga tggatgatag at - #acatgctt        360                                                                          - acagatgcac acacaaacgt aaatggtatn aaaaatngga tncactcttg ta - #nggttgtt        420                                                                          #          426                                                                - <210> SEQ ID NO 110                                                         <211> LENGTH: 350                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 110                                                         - aggttaaggc tgcagtgagc catgttcatg ccactgcact tcactctgag tg - #acaaattg         60                                                                          - agaccttgtc tcagaaagaa agaaagaaag aaagaaagaa agaaagaaag aa - #ngaaagaa        120                                                                          - agaaagtaag aaaaagagag ggaaagaaag agaaanagna aanaaatagt ag - #caactgtt        180                                                                          - attgtaagac atctccacac accagagaag ttaattttaa ttttaacatg tt - #aagaacag        240                                                                          - agagaagcca acatgtccac cttaggctga cggtttgttt atttgtgttg tt - #gctggtag        300                                                                          #             350tttaaa gtagcttatc caatacttca ttaacaattt                      - <210> SEQ ID NO 111                                                         <211> LENGTH: 528                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 111                                                         - ctaccaatca tagtggaaag caaagacaga gcaaggcatc tcacatggct ag - #agcaggag         60                                                                          - caagagaaag ataggggagc ttgtagatgg tctgttatgg gacttttctc ag - #tctccata        120                                                                          - aatatgtgag tcaattcccc aagtgaattg ccttctatct atctatctat ct - #gtctgtct        180                                                                          - gtctgtctgt ctgtctatct atctatatct atctatctat catctatcta tc - #tatctatc        240                                                                          - tatctatcta tctatctatc tatcgtctat ctatccagtc tatctacctc ct - #attagtct        300                                                                          - gtctctggag aacattgact aatacaacat ctttaatata tcacagttta at - #ttcaagtt        360                                                                          - atatcatacc acttcataca ttatataaaa ccttacagtg tttctccctt ct - #cagtgttt        420                                                                          - atggctagta attttttact gggtgccaga cactaatttt tattttgcta ag - #tggtgaat        480                                                                          #               528aaaa tatttttgag tgttgatctg ggtaaagt                        - <210> SEQ ID NO 112                                                         <211> LENGTH: 194                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 112                                                         - ctactgagtt tctgttatag tgttttttaa tatatatata gtattatata ta - #tagtgtta         60                                                                          - tatatatata gtgttttaga tagatagata ggtagataga tagatagata ga - #tagataga        120                                                                          - tagatagata gatagataga tatagtgaca ctctccttaa cccagatgga ct - #ccttgtcc        180                                                                          #    194                                                                      - <210> SEQ ID NO 113                                                         <211> LENGTH: 320                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 113                                                         - cacttgaacc cgggaggtgg aggttgcact ccagcctttg caacagagca ag - #acttcatc         60                                                                          - tgaaagatag aaagatgaaa gaaagaaaga aagaaagaaa gaaagagtaa aa - #gaaaaaaa        120                                                                          - ttaaaatttt agggggaaaa ttttctaatt tttgaacatg cactaaaatg at - #tttcagag        180                                                                          - aaaaccaagt gttattttct aatctgcatg gcattattaa agatgtttac tc - #atcttcct        240                                                                          - tggggctagg catcccattc ctgcaggaag tcttgtggtt aggcggtggc tg - #tggctctg        300                                                                          #320               caga                                                       - <210> SEQ ID NO 114                                                         <211> LENGTH: 330                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 114                                                         - gggatttccc tatggattgg aagtggggcg tgaaatagag gagtcagggg tc - #actctggg         60                                                                          - gatttggcct ggagcagctg gaagatggag tggctgttaa ttcatgtagg ga - #aggctgtg        120                                                                          - ggaagaagag gtttaggaga caaggatagc agttcattta tttatttatt ta - #tttattta        180                                                                          - tttatttatt tatttattta gagatgtagt ctcattcttt cgccaggctg ga - #gtgcagtg        240                                                                          - gcgcgatctt ggctcactgc aacctccacc tcccaggctc aagcgattct ct - #tgcctcag        300                                                                          #          330     gtag ctgggactac                                            - <210> SEQ ID NO 115                                                         <211> LENGTH: 192                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 115                                                         - gccccatagg ttttgaactc acagattaaa ctgtaaccaa aataaaatta gg - #catattta         60                                                                          - caagctagtt tctttctttc ttttttctct ttctttcttt ctttctttct tt - #ctttcttt        120                                                                          - ctttctttct ttctttcttt ctccttcctt cctttcttcc tttctttttt gc - #tggcaatt        180                                                                          #      192                                                                    - <210> SEQ ID NO 116                                                         <211> LENGTH: 320                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 116                                                         - aggtatactt ttctctccag aatagttaga tgtaggtata ccactttgat gt - #tgacacta         60                                                                          - gtttacctag aacttatctt ctgtaaatct gtctctattt ccatctctgt ct - #ccatcttt        120                                                                          - gtctctatct ctatctgtct atctctatct atctatctat ctatctatct at - #ctatctat        180                                                                          - ctatctatct atctaaagca aattcatgcc cttctcctat ttattgaatc ga - #gaccatag        240                                                                          - acaggggtga gagaaagaat ttggcaggaa tggggatgtg tattatctgt gg - #cataagga        300                                                                          #320               gttc                                                       - <210> SEQ ID NO 117                                                         <211> LENGTH: 300                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 117                                                         - gcccttccca ggctctagca gcagctcatg gtggggggtc ctgggcaaat ag - #ggggcaaa         60                                                                          - attcaaaggg tatctgggct ctggggtgat tcccattggc ctgttcctcc ct - #tatttccc        120                                                                          - tcattcattc attcattcat tcattcattc attcattcac catggagtct gt - #gttccctg        180                                                                          - tgacctgcac tcggaagccc tgtgtacagg ggactgtgtg ggccaggctg ga - #taatcggg        240                                                                          - agcttttcag cccacaggag gggtcttcgg tgcctccttg ggcactcaga ac - #cttgggct        300                                                                          - <210> SEQ ID NO 118                                                         <211> LENGTH: 300                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 118                                                         - agcacccaga accgtcgact ggcacagaac aggcacttag ggaaccctca ct - #gaatgaat         60                                                                          - gaatgaatga atgaatgaat gaatgaatga atgaatgttt gggcaaataa ac - #gctgacaa        120                                                                          - ggacagaagg gcctagcggg aagggaacag gagtaagacc agcgcacagc cc - #gacttgtg        180                                                                          - ttcagaagac ctgggattgg acctgaggag ttcaattttg gatgaatctc tt - #aattaacc        240                                                                          - tgtgtggttc ccagttcctc ccctgagcgc ccaggacagt agagtcaacc tc - #acgtttga        300                                                                          - <210> SEQ ID NO 119                                                         <211> LENGTH: 143                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 119                                                         - gtgagttagc cgtttagcga tatatacata ttatgaaaca ttattattat ta - #ttattatt         60                                                                          - attattatta ttattattat tattattatt tgagacggac tctcgctctg tc - #gcccaggc        120                                                                          #               143cgat ctg                                                   - <210> SEQ ID NO 120                                                         <211> LENGTH: 279                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 120                                                         - ctattcattc aatcatacac ccatatctgt ctgtctgtct atctatctat ct - #atctatct         60                                                                          - atctatctat ctatctgcct atctgcctgc ctacctatcc ctctatggca at - #tgcttgca        120                                                                          - accagggaga ttttattccc aggagatatt tggctatgtg tgacaacaat tt - #ttttggtt        180                                                                          - gtcacaaatg ggatgaatgt tactggcatc tggtgggtgg agcccagaga tg - #ctgctcaa        240                                                                          #   279            agac agacccacca caaagaatc                                  - <210> SEQ ID NO 121                                                         <211> LENGTH: 263                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 121                                                         - tcattaatct agcttttaaa aacaactaat ttgatttcaa gtgtttgtta tt - #taaaagcc         60                                                                          - aagaaggaaa acaaattttt ttcttgtatc accatttatt tattattatt at - #tattatta        120                                                                          - ttattattat tattattatt attattattt actaaggaat gggattggta gg - #tttaatga        180                                                                          - tccctctgtt ttgacttctt tgagatattt ccagactact ttccactttg ac - #tgtaggaa        240                                                                          #               263tggg tct                                                   - <210> SEQ ID NO 122                                                         <211> LENGTH: 131                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 122                                                         - gtggtcttct acttgtgtca atacagatag atagatagat agatagatag at - #agatagat         60                                                                          - agatagatag atagatagat agatatgtat gtcttttcta tgagacatac ct - #catttttt        120                                                                          #      131                                                                    - <210> SEQ ID NO 123                                                         <211> LENGTH: 372                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 123                                                         - catgngcccc caaagcgnag tnaacttnac ccagtgtcac aaaatggcct tt - #nacgaatt         60                                                                          - actcctccat tgtccaccca tctnatactc actgtctgga tttcttggtt at - #agtaaatc        120                                                                          - tagatctatc tatctatcta tctatctatc tatctatcta tctatctatc ta - #tctgtgta        180                                                                          - tctctctacc agctttttta acttgtcctt aattgttcaa tttatatata at - #gagaaaat        240                                                                          - ggttatantt tcctgagngc ngnnttacca tagtagngca aangagttgc ag - #cancaggg        300                                                                          - ncaacattgn cacttctngg ttattccncc aatgtttncc ntttnccnta aa - #tttnaatt        360                                                                          #      372                                                                    - <210> SEQ ID NO 124                                                         <211> LENGTH: 240                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 124                                                         - agctacagca aacttcatgt gacaaaagcc acacccataa ctttttncct ct - #agatagac         60                                                                          - agatagatga tagatagata gatagataga tagatagata gatagataga ta - #gatagata        120                                                                          - gatatagatt ctctttctct gcattctcat ctatatttct gtctttctct ta - #attatggg        180                                                                          - taactcttag cctgccaggc taccatggaa agacaacctt tattcctctt tt - #ctcctggc        240                                                                          - <210> SEQ ID NO 125                                                         <211> LENGTH: 325                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 125                                                         - gtgggaggaa gccagtggat ttggaaacag aaatggcttg gccttgcctg cc - #tgcctgcc         60                                                                          - tgcctgcctt ccttccttcc ttccttcctt ccttccttcc ttccttcctt cc - #ctcctgca        120                                                                          - atcctttaac ttactgaata actcattatt atgggccncc tgcaggtacc at - #gctaggta        180                                                                          - ctagggatgt aggcatgaac actgacaagg gcctctggga ctggcattct gg - #taggaaaa        240                                                                          - ggggtgagac agggaagaag ccagcaaatg tatcaacaag aaacagttct aa - #gtgctagg        300                                                                          #              325 gatg tcaca                                                 - <210> SEQ ID NO 126                                                         <211> LENGTH: 269                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 126                                                         - aaagctataa ttgtaccact gcactccagc ctgggcaaca gaataagatt ct - #gttgaagg         60                                                                          - aaagaaggta ggaaggaagg aaggaaggaa ggaaggaagg aaggaaggaa gg - #aaggagag        120                                                                          - aggtagaaag agagaagatt tttattcggg taatgggtgc accaaaatat ca - #gaaatcac        180                                                                          - tgctaaagaa cttattcatg taaccaacac cacctgttcc ttaaaaacct at - #tgaaataa        240                                                                          #           269    agaa agaggnnga                                             - <210> SEQ ID NO 127                                                         <211> LENGTH: 377                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 127                                                         - aaagtcttca aagcatcctg aagttggtct taagccagca ttcttaaaac tc - #taaggagg         60                                                                          - caacaaaaga tttaaacagt gtacagcaaa tggtgactct gaaaccagag tt - #gtttcact        120                                                                          - gctcactgcc accccgagat tgatttgcca tgatagatgg cttcctaggc tc - #aattaggt        180                                                                          - tcttaattat ggagatagtt atatttactt ctgtcacagg gctgatgagg tg - #aaatattt        240                                                                          - gcaaaacaat ctatctatat ctatctatat ctatctatct atctatctat ct - #atctatct        300                                                                          - atctatctat ctatcatctt atatgtgttg ttgttgaggt tgtttgagat at - #cccccagg        360                                                                          #  377             t                                                          - <210> SEQ ID NO 128                                                         <211> LENGTH: 344                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 128                                                         - tttggactgg aacttacact gttggttctc cttgttctca gacctttgaa ct - #cagactga         60                                                                          - aaccacatac tcagcactcc tgggtctcta gcttgccaag tgcccaagtg ca - #gatcttgg        120                                                                          - gacttctcgg tgccgttatt gtgtgagtca attccttgtt ataaaattat at - #atacatat        180                                                                          - atttgtagat ggatagaaga tgatagatag atagataggt agatagatag at - #agatagat        240                                                                          - agatagatag atagatagat tctgtttctc tggagaactc taatgcagtt gc - #ccacactc        300                                                                          #344               attt cattgataac ttaccttctg aaat                            - <210> SEQ ID NO 129                                                         <211> LENGTH: 372                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 129                                                         - aaagctacat ccaaattagg taggtagaca aataggtagg taggtagaca ga - #cagacaga         60                                                                          - ctagatagat ggacagacta gatagataga tacgtacata cataagatag at - #agatagat        120                                                                          - agatagatag atagatagat agatagatag atagatagat agagacagat tt - #aaaatatt        180                                                                          - tgggacattt tagtttcttt gtcactcttt gaactggaac tataaaaaat ac - #tcttttac        240                                                                          - tatcacaaga ggatagagga cctaatataa tgctactgct gtgtctcaac ag - #tgacagcc        300                                                                          - aggtacaaag gttaccatta cttccctttg ggctctgagt gtgtcttgcc tg - #cagccacc        360                                                                          #      372                                                                    - <210> SEQ ID NO 130                                                         <211> LENGTH: 355                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 130                                                         - ttacctaaat ctgtctcaga ccatacctaa atctctctct ctctttctct ct - #gtctctcc         60                                                                          - ctctccctct cttacagggc agttgtttat agaatatatc tcaatttgag tt - #tgatgttt        120                                                                          - ttgagagaca gaatatctat ctgtctgtct atctatccat ccatccatct at - #catctatt        180                                                                          - tattatctat ctatctatct atctatctat ctatctatct atctatcctg ct - #tttctaga        240                                                                          - gaacacagac taatgtaggt gataactagg atcccttccc cactaagaat ng - #ttcagggc        300                                                                          - cctgcacccc agaggaggaa cctatttcct ttctttcccc tgggatccac tg - #ctt             355                                                                          - <210> SEQ ID NO 131                                                         <211> LENGTH: 320                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 131                                                         - taactgtaat atttgctaca acgttaataa ccaaattgtt tatgaggtgg tg - #tactacca         60                                                                          - tatttgaaca tgtgctcaaa tattgttaaa gagacacaat taaagaaaga at - #gacccttg        120                                                                          - gaattttatt taattttatt tatttattta tttatttatt tatttattta tt - #tagagaca        180                                                                          - gagtcttgct ctgtcgccca gcctagagtg caatggcatg atcttggctc ac - #tgcaattt        240                                                                          - ttgcctcccg ggttcaagca attctccttc ctcagccttc caagtagctg gg - #attacagg        300                                                                          #320               cgct                                                       - <210> SEQ ID NO 132                                                         <211> LENGTH: 159                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 132                                                         - ttggagtcgc aagctgaact agcgttttct tttcttttcc tttcttttct tt - #tcttttct         60                                                                          - tttcttttct tttcttcttt tcaagacagg ttctcactct gtcactcagg ct - #agagtgca        120                                                                          #   159            tcac tgcagcctca acttcctgg                                  - <210> SEQ ID NO 133                                                         <211> LENGTH: 229                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 133                                                         - aacaggatca atggatgcat aggtagatag atagatagat agatagatag at - #agatagat         60                                                                          - agatagatag atagatagac agacagacag acagacagac agatgagagg gg - #atttatta        120                                                                          - gaggaattag ctcaagtgat atggaggctg aaaaatctca tgacagtcca tc - #tgcaagct        180                                                                          #              229ctagg agcatggctc agtccaggtc taaaagcca                       - <210> SEQ ID NO 134                                                         <211> LENGTH: 379                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 134                                                         - agctcaatat aacttcacag attgaacaca tccatgtaac cagcacccag at - #taagaaac         60                                                                          - agagcatgac tagcacaatc tcatgcttcc ttttagacac tacagttgac tc - #ttaaataa        120                                                                          - tttggggatt aggggtgcag ttgaaaatcc aagtataatt ttgtctccct ga - #aaatgtaa        180                                                                          - ctagtaatag cctactgttg actggaagcc ttactgactt actacataac ga - #cacacaca        240                                                                          - cacacacaca cacacacaca cacacacaca cacacacata tatatatttt ga - #gatgcagt        300                                                                          - cttgctctgt tgcccaggct ggagtncagt ggcacgatct cggctcactg ca - #acctccgc        360                                                                          #379               gtt                                                        - <210> SEQ ID NO 135                                                         <211> LENGTH: 387                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                  - <400> SEQUENCE: 135                                                         - gaattataac cgtaactgat tcatagcagc acttgccaaa ttctattttg tg - #gaaaaata         60                                                                          - ttctgggaag atattaacaa tgtnacacac acacacacac acacacacac ac - #acacacac        120                                                                          - gatgtacatg gttttaaaaa tgtcaacagg ttcctttgct ggaggaattc cc - #agtgtctt        180                                                                          - tgttatagga atcttcactg ggaataaagt gataatagca gtggtaatgg aa - #atgtttta        240                                                                          - ttgactgctt aaactgaagt canacaagca ttatctcact ttttttataa ac - #attattta        300                                                                          - attctcaaaa cagacctgtg cagtaggtac aattatgtgg tacacagatg ag - #aaactgag        360                                                                          #            387   ataa cccagct                                               __________________________________________________________________________

What is claimed is:
 1. A method for multiplexing the identification ofmore than one DNA tandem nucleotide repeat region from more than one DNAtandem nucleotide repeat loci, comprising:obtaining more than onenucleic acid extension product by extending one or more primerscomplementary to sequences flanking the DNA tandem nucleotide repeatregion, and; determining the masses of more than one nucleic acidextension product simultaneously by mass spectrometry,wherein thenucleic acid extension products have overlapping allelic mass ranges. 2.The method of claim 1, wherein a 3' end of one or more primersimmediately flanks a DNA tandem nucleotide repeat region.
 3. The methodof claim 1, wherein one or more primers comprise a sequencecomplementary to up to one tandem repeat of the DNA tandem nucleotiderepeat locus.
 4. The method of claim 3, wherein one or more primerscomprise a sequence complementary to up to two tandem repeats of the DNAtandem nucleotide repeat locus.
 5. The method of claim 4, wherein one ormore primers comprise a sequence complementary to up to three tandemrepeats of the DNA tandem nucleotide repeat locus.
 6. The method ofclaim 1, wherein the extension of at least one primer is terminatedusing a chain termination reagent.
 7. The method of claim 6, wherein thechain termination reagent is a dideoxynucleotide triphospate.
 8. Themethod of claim 6, wherein at least one target nucleic acid extensionproduct contains a mass modifying group.
 9. The method of claim 8,wherein the mass modifying group comprises a mass modified nucleotide.10. The method of claim 8, wherein the mass modifying group comprises anonstandard deoxyribonucleotide.
 11. The method of claim 1, wherein atleast one primer comprises a cleavable site and wherein the cleavablesite comprises a recognition site for a restriction endonuclease, anexonuclease blocking site, or a chemically cleavable site.
 12. Themethod of claim 11, wherein the cleavable site comprises a recognitionsite for a restriction endonuclease.
 13. The method of claim 11, whereinthe cleavable site comprises an exonuclease blocking site.
 14. Themethod of claim 11, wherein the cleavable site comprises a chemicallycleavable site.
 15. The method of claim 8, wherein the mass modifyinggroup is incorporated during extension of the nucleic acid extensionproduct.
 16. The method of claim 8, wherein the mass modifying group isincorporated after extension of the nucleic acid extension product. 17.A method for multiplexing the identification of more than one DNA tandemnucleotide repeat region from more than one DNA tandem nucleotide repeatloci, comprising:obtaining more than one nucleic acid amplificationproduct by amplifying two or more primers complementary to sequencesflanking the DNA tandem nucleotide repeat region; and determining themasses of more than one nucleic acid amplification productsimultaneously by mass spectrometry,wherein the nucleic acid extensionproducts have overlapping allelic mass ranges.
 18. The method of claim17, wherein a 3' end of one or more primers immediately flanks a DNAtandem nucleotide repeat region.
 19. The method of claim 17, wherein oneor more primers comprise a sequence complementary to up to one tandemrepeat of the DNA tandem nucleotide repeat locus.
 20. The method ofclaim 19, wherein one or more primers comprise a sequence complementaryto up to two tandem repeats of the DNA tandem nucleotide repeat locus.21. The method of claim 20, wherein one or more primers comprise asequence complementary to up to three tandem repeats of the DNA tandemnucleotide repeat locus.
 22. The method of claim 17, wherein at leastone target nucleic acid amplification product contains a mass modifyinggroup.
 23. The method of claim 22, wherein the mass modifying groupcomprises a mass modified nucleotide.
 24. The method of claim 22,wherein the mass modifying group comprises a nonstandarddeoxyribonucleotide.
 25. The method of claim 17, wherein at least oneprimer comprises a cleavable site and wherein the cleavable sitecomprises a recognition site for a restriction endonuclease, anexonuclease blocking site, or a chemically cleavable site.
 26. Themethod of claim 25, wherein the cleavable site comprises a recognitionsite for a restriction endonuclease.
 27. The method of claim 25, whereinthe cleavable site comprises an exonuclease blocking site.
 28. Themethod of claim 25, wherein the cleavable site comprises a chemicallycleavable site.
 29. The method of claim 22, wherein the mass modifyinggroup is incorporated during amplification.
 30. The method of claim 22,wherein the mass modifying group is incorporated after amplification.